Upload
vivek-ananth-r-p
View
220
Download
0
Embed Size (px)
Citation preview
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 128
YEASTBOOK
CELL STRUCTURE amp TRAFFICKING
Secretory Protein Biogenesis and Traf1047297c in the EarlySecretory Pathway
Charles K Barlowe and Elizabeth A Millerdagger1
Department of Biochemistry Dartmouth Medical School Hanover New Hampshire 03755 and ySchool of Biological Sciences Columbia University
New York New York 10027
ABSTRACT The secretory pathway is responsible for the synthesis folding and delivery of a diverse array of cellular proteins Secretory
protein synthesis begins in the endoplasmic reticulum (ER) which is charged with the tasks of correctly integrating nascent proteins and
ensuring correct post-translational modi1047297cation and folding Once ready for forward traf1047297c proteins are captured into ER-derived
transport vesicles that form through the action of the COPII coat COPII-coated vesicles are delivered to the early Golgi via distinct
tethering and fusion machineries Escaped ER residents and other cycling transport machinery components are returned to the ER via
COPI-coated vesicles which undergo similar tethering and fusion reactions Ultimately organelle structure function and cell
homeostasis are maintained by modulating protein and lipid 1047298ux through the early secretory pathway In the last decade structural and
mechanistic studies have added greatly to the strong foundation of yeast genetics on which this 1047297eld was built Here we discuss the key
players that mediate secretory protein biogenesis and traf1047297cking highlighting recent advances that have deepened our understanding
of the complexity of this conserved and essential process
TABLE OF CONTENTS
Abstract 383Introduction 384
Expanding Methodologies From a Parts List to Mechanisms and Back to More Parts 384
Classic screens lay the groundwork in vitro reconstitution de1047297nes mechanism 384
Dynamics and organization revealed by live cell imaging 385
New technologies yield new players and de1047297 ne interplay between pathways 385
Secretory Protein Translocation and Biogenesis 386
Polypeptide targeting and translocation 386
Maturation of secretory proteins in the ER signal sequence processing 388
Maturation of secretory proteins in the ER protein glycosylation 388
Maturation of secretory proteins in the ER glycosylphosphatidylinositol anchor addition 389
Maturation of secretory proteins in the ER disul 1047297
de bond formation 389Glucosidase mannosidase trimming and protein folding 390
Control of ER homeostasis by the Unfolded Protein Response 391
Continued
Copyright copy 2013 by the Genetics Society of Americadoi 101534genetics112142810Manuscript received June 14 2012 accepted for publication September 25 20121Corresponding author School of Biological Sciences Columbia University 1212 Amsterdam Ave MC2456 New York NY 10027 Email em2282columbiaedu
Genetics Vol 193 383ndash410 February 2013 383
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 228
CONTENTS continued
Transport From the ER Sculpting and Populating a COPII Vesicle 391
Structure and assembly of the COPII coat 392
Cargo capture stochastic sampling vs direct and indirect selection 393
Regulation of COPII function GTPase modulation coat modi 1047297 cation 394
Higher-order organization of vesicle formation 395
Vesicle Delivery to the Golgi 395
Vesicle tethering 395
SNARE protein-dependent membrane fusion 396
A concerted model for COPII vesicle tethering and fusion 397
Traf1047297c Within the Golgi 397
Transport through the Golgi complex 397
Lipid requirements for Golgi transport 398
The Return Journey Retrograde Traf1047297c via
COPI Vesicles 398
Composition and structure of the COPI coat 399
Cargo capture sorting signals cargo adaptors and coat stimulators 400
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion 401
Perspectives 401
LIKE all eukaryotes yeast cells segregate various physio-
logical functions into distinct subcellular compartments
A key challenge is thus ensuring that appropriate proteins
are delivered to the correct subcellular destination a process
that is driven by discrete sorting signals that reside in the
proteins themselves Perhaps the most prevalent type of sort-
ing signal is that directing a protein to the secretory pathway
which handles the various proteins that are destined for the
extracellular environment or retention in the internal endo-
membrane system Approximately one-third of the yeast pro-
teome enters the secretory pathway Protein secretion is not
only essential for cellular function but also provides the
driving force for cell growth via delivery of newly synthe-
sized lipid and protein that permits cell expansion Secretory
proteins enter this set of interconnected organelles at the
endoplasmic reticulum (ER) which regulates protein trans-
lation protein translocation across the membrane protein
folding and post-translational modi1047297cation protein quality
control and forward traf 1047297c of suitable cargo molecules (both
lipid and protein) Once contained within the secretory path-
way proteins are ferried between compartments via trans-port vesicles that bud off from one donor compartment to
fuse with a downstream acceptor compartment thereby
mediating directional traf 1047297c of both lipid and protein The
forward-moving or anterograde pathway is balanced by
a reverse or retrograde pathway that returns escaped resi-
dent proteins and maintains the homeostasis of individual
organelles Early yeast screens pioneered the genetic dissec-
tion of the eukaryotic secretory pathway and were rapidly
followed by biochemical approaches that permitted the mo-
lecular dissection of individual processes of protein biogen-
esis and traf 1047297c Here we discuss the methodologies that
have yielded great insight into the conserved processes that
drive protein secretion in all eukaryotes and describe the
fundamental processes that act to ensure ef 1047297cient and ac-
curate protein secretion The reader is also referred to earlier
comprehensive reviews on these topics (Kaiser et al 1997
Lee et al 2004) as we focus our coverage on more recent
advances
Expanding Methodologies From a Parts Listto Mechanisms and Back to More Parts
Classic screens lay the groundwork in vitro reconstitutionde1047297 nes mechanism
There is no doubt that early seminal yeast genetics ap-
proaches laid the foundation upon which our understand-
ing of protein secretion is built From the original Novick
and Schekman screens that identi1047297ed a host of secretion-
defective ( sec) mutants (Novick and Schekman 1979 Novick
et al 1980) to additional more targeted approaches fromthe Schekman (more secs Deshaies and Schekman 1987
Wuestehube et al 1996) Gallwitz ( ypt Gallwitz et al 1983)
Ferro-Novick (bet Newman and Ferro-Novick 1987) Jones
( pep Jones 1977) Stevens ( vps Rothman et al 1989) and
Emr ( vps Bankaitis et al 1986) labs that expanded the rep-
ertoire of mutants with defects in secretory protein and
membrane biosynthesis the 1047297eld has been blessed with an
abundance of reagents that permitted the characterization
of each branch of the secretory pathway (Schekman and
384 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 328
Novick 2004) Many of these processes are essential con-
served and have direct relevance to issues of human health
yet yeast genetics approaches remain at the forefront in
deciphering molecular mechanisms unraveling cellular re-
dundancy and complexity and appreciating the cross-talk
between different branches of the pathway The strength of
yeast as a model system to probe this complexity lies in the
combination of facile genetics and robust biochemistry that
are afforded by this remarkable organism Indeed the 1047297
eldhas a long history of capitalizing on yeast mutants to inform
biochemical reconstitution approaches that in turn inform
new genetic screening approaches
The most pertinent example of the strength of this
approach is the mechanistic description of the COPII coat
proteins that drive vesicle formation from the endoplasmic
reticulum Classic epistasis analyses of the Novick and
Schekman sec mutants (Novick et al 1980) placed the early
sec genes in order within the secretory pathway sec12
sec13 sec16 and sec23 mutants blocked formation of trans-
port vesicles and induced proliferation of the ER whereas
sec17 sec18 and sec22 mutants blocked vesicle fusion and
caused accumulation of vesicles (Novick et al 1981 Kaiserand Schekman 1990) The subsequent development of
in vitro assays relied in part on the use of these mutants in
biochemical complementation assays (Baker et al 1988
Ruohola et al 1988) Recapitulation of ER ndashGolgi traf 1047297c in
permeabilized yeast cells was perturbed in sec23 mutants
but could be restored by incubation with cytosol prepared
from wild-type cells placing Sec23 as a soluble factor re-
quired for transport vesicle formation (Baker et al 1988)
Further re1047297nement of these in vitro transport assays permit-
ted the dissection of different transport stages (Rexach and
Schekman 1991) and allowed the biochemical characteriza-
tion of the COPII coat proteins (Barlowe et al 1994) that
generate transport intermediates and the membrane-bound
and cytosolic factors required for tethering and fusion steps
that consume vesicles at the Golgi membrane (Barlowe
1997 Cao et al 1998) Further mechanistic dissection came
from even more re1047297ned reconstitution systems that permit-
ted the identi1047297cation of the minimal machinery required to
generate COPII vesicles from synthetic liposomes (Matsuoka
et al 1998ab) and de1047297ned the dynamics of individual
events using real-time assays (Antonny et al 2001)
Similar reconstitution of the COPI-mediated GolgindashER
retrograde pathway in yeast lagged somewhat behind in
part due to equivalent biochemical experiments that were
under way in mammalian cells (Balch et al 1984 Waterset al 1991) Furthermore due to rapid perturbation in for-
ward (ER ndashGolgi) traf 1047297c when the retrograde pathway is
blocked for some time there was confusion over the direc-
tionality of COPI-mediated events (Gaynor and Emr 1997)
Despite these dif 1047297culties in vitro reconstitution of COPI-
coated vesicle formation was ultimately achieved (Spang
and Schekman 1998) and has been similarly dissected
in minimal systems using synthetic liposomes (Spang et al
1998)
In contrast to the genetics-informed biochemical ap-
proaches described above minimal reconstitution of the
membrane fusion events that drive vesicle consumption took
a slightly different path Armed with the knowledge that
fusion is driven by proteins known as SNAREs (soluble N-
ethylmaleimide-sensitive factor attachment protein recep-
tors) and with the full description of yeast SNAREs in hand
from computational analyses of the yeast genome Rothman
and colleagues established liposome-based assays that dem-onstrated compartment speci1047297city of different SNARE pairs
(McNew et al 2000) That this biochemical approach largely
recapitulated known pathways previously de1047297ned by ge-
netic means serves to highlight the success of mutually in-
formed genetic and biochemical approaches to fully dissect
the molecular mechanisms of budding and fusion events
Dynamics and organization revealed by live cell imaging
With budding and fusion machineries well described in
minimal systems it became apparent that there were still
pieces of the puzzle missing including the roles of some
essential proteins (eg Sec16 Espenshade et al 1995) that
remained unexplained in terms of functionality Further-more some of the more pressing mechanistic questions
could not be answered by biochemical means For example
the mode of protein and lipid traf 1047297c through the Golgi
remained controversial did COPI vesicles mediate forward
traf 1047297c or did proteins proceed through the Golgi by a process
of maturation of individual cisternae These questions were
addressed in part by the Glick and Nakano labs using high-
resolution time-lapse imaging of living yeast cells (Losev
et al 2006 Matsuura-Tokita et al 2006) Such experiments
de1047297ned discrete sites of vesicle formation known as transi-
tional ER (tER) or ER exit sites (ERES) that are dynamic in
nature can form de novo but also fuse with each other and
have clear relationships with downstream Golgi elements
(Bevis et al 2002 Shindiapina and Barlowe 2010) Further-
more imaging of distinct Golgi elements lent support for the
cisternal maturation model of protein secretion although
direct imaging of cargo molecules remains to be fully dem-
onstrated Recent advances in superresolution imaging hold
great promise in further understanding the nature of these
subdomains and their relationships with distinct protein
machineries and membrane compartments although some
limitations will still apply especially with respect to the
problem of detecting transient cargo molecules that are
in 1047298ux through the system
New technologies yield new players and de1047297 ne interplay between pathways
Since the yeast community entered the postgenomic world
a host of new tools has opened up many new approaches
the haploid deletion collection represents an accessible
large-scale analysis platform for novel screens (Tong
et al 2001) the GFP- (Huh et al 2003) and TAP-tagged
(Ghaemmaghami et al 2003) fusion databases documented
the localization and abundance of many gene products and
Early Events in Protein Secretion 385
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 428
microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation
(Travers et al 2000) These new tools are being used with
remarkable imagination often capitalizing on the facile na-
ture of yeast genetics to de1047297ne the interplay between related
pathways in exciting ways For example microarray analysis
of the changes in gene expression that occur upon induction
of ER stress via the unfolded protein response (UPR) iden-
ti1047297ed upregulation of machineries involved in ER-associated
degradation (ERAD) ultimately leading to the appreciation
that these discrete pathways are intimately coordinated to
manage the burden of protein within the ER (Travers et al
2000) A second example derives from the development of
synthetic genetic array (SGA) technology which allows the
rapid generation of haploid double mutant strains (Tong
et al 2001) Although the piecemeal application of this tech-
nology was informative for individual genes the broader
application to an entire pathway was revolutionary in terms
of being able to de1047297ne novel functions based on shared
genetic 1047297ngerprints The 1047297rst so-called epistatic miniarray
pro1047297le (E-MAP) made pairwise double mutations among
almost 500 early secretory pathway components quantify-
ing the phenotypic cost of combined mutations (Schuldiner
et al 2005) Analysis of the shared patterns of genetic inter-
actions revealed (perhaps not surprisingly) that components
in common pathways shared similar pro1047297les which allowedthe assignation of novel functions to previously uncharacter-
ized and enigmatic proteins An elaboration on the E-MAP
approach made elegant use of a 1047298uorescent reporter system
to 1047297rst assess the UPR state of individual strains in the
genomic deletion collection and then to probe how UPR
activation changes in double mutant backgrounds yielding
a more subtle understanding of genetic interactions than
gross life and death dichotomies which usually form the
basis of synthetic interactions (Jonikas et al 2009) With
further development of such reporters on cell status thisarea of cross-talk between pathways will become more
and more integrated allowing a detailed picture of cellu-
lar physiology However as these new technologies yield
new functional clues to previously uncharacterized genes
we need to continue to use and develop biochemical tools
that allow true mechanistic insight Again the strength of
the yeast system is the use of both genetic and biochemical
tools to mutually inform new discoveries
Secretory Protein Translocation and Biogenesis
Polypeptide targeting and translocation
The 1047297rst step in biogenesis of most secretory proteins is
signal sequence-directed translocation of the polypeptide
into the ER Both cotranslational and post-translational
mechanisms operate in yeast to target diverse sets of soluble
and integral membrane secretory proteins to the ER (Figure
1) The cotranslational translocation process is initiated
when a hydrophobic signal sequence or transmembrane
sequence is translated and recognized by the signal-recognition
particle (SRP) for targeting to the SRP receptor at ER trans-
location sites (Figure 1a) In the case of post-translational
translocation cytosolic chaperones play a critical role in
binding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-
location competent state until delivery to the ER membrane
(Figure 1b) Progress on identi1047297cation and characterization
of the translocation machinery will be described in turn
below as the start of a continuum of events in biogenesis
of secretory proteins
Genetic approaches in yeast uncovered key components
in both the co- and post-translational translocation path-
ways Appending a signal sequence to the cytosolic enzyme
Figure 1 Membrane transloca-
tion of secretory proteins Three
well-characterized pathways oper-
ate to deliver secretory proteins
to the ER for membrane trans-
location (A) The signal recogni-
tion particle (SRP) recognizes a
hydrophobic signal sequence or
transmembrane segment during
protein translation followed by
targeting of the ribosomendash
nascentchain complex to the SRP receptor
for cotranslational membrane in-
sertion (B) Post-translational inser-
tion of secretory proteins depends
on cytosolic Hsp70 ATPases such
as Ssa1 to maintain the nascent
secretory protein in an unfolded
translocation competent state until delivery to the Sec63 complex formed by Sec62Sec63Sec71Sec72 The Sec61 complex forms an aqueous
channel for both post- and cotranslational polypeptide translocation Kar2 a luminal Hsp70 ATPase facilitates directed movement and folding
of nascent polypeptides (C) In GET-mediated insertion of C-terminal tail-anchored proteins the Sgt2ndashGet4ndashGet5 complex targets nascent
polypeptides to Get3 for Get1Get2 dependent translocation Tail-anchored proteins are integrated into the membrane in Sec61-independent
pathway
386 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 528
encoded by HIS4 targets this enzyme to the ER where it
cannot function and produces histidine auxotrophy A ge-
netic selection for mutants that are partially defective in
translocation of this signal peptide-bearing fusion protein
and therefore restore histidine prototrophy was used to
identify conditional mutations in three essential genes
SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987
Rothblatt et al 1989) Sequencing indicated that all three
genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-
membrane segments and striking sequence identity with the
Escherichia coli translocation protein SecY (Stirling et al
1992 Jungnickel et al 1994) Similar genetic selection
approaches using the HIS4 gene product fused to integral
membrane proteins identi1047297ed SEC65 which encodes a com-
ponent of the SRP (Stirling and Hewitt 1992 Stirling et al
1992) as well as mutations in SEC71 and SEC72 (Green
et al 1992)
Concurrent with these genetic approaches cell-free
reconstitution assays that measured post-translational
translocation of radiolabeled pre-pro-a-factor into yeast
microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt
and Meyer 1986) Fractionation of cytosolic components re-
quired in the cell-free assay revealed that Hsp70 ATPases
stimulated post-translational translocation (Chirico et al
1988) Yeast express a partially redundant family of cyto-
solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-
lectively essential An in vivo test for Hsp70 function in
translocation was demonstrated when conditional expres-
sion of SSA1 in the background of the multiple ssa D strain
resulted in accumulation of unprocessed secretory proteins
as Ssa1 was depleted (Deshaies et al 1988) ATPase activity
of Hsp70 family members is often stimulated by a corre-
sponding Hsp40 Dna J partner and in the case of poly-
peptide translocation in yeast the YDJ1 gene encodes
a farnsylated DnaJ homolog that functions in ER transloca-
tion (Caplan et al 1992) Ydj1 has been shown to directly
regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer
et al 1995) and structural studies indicate that Ydj1 binds to
three- to four-residue hydrophobic stretches in nonnative
proteins that are then presented to Hsp70 proteins such as
Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-
ments connect YDJ1 to translocation components in addi-
tion to multiple other cellular pathways presumably due to
action on a subset of secretory proteins (Becker et al 1996
Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the
multispanning Sec61 forms an aqueous channel for polypep-
tide translocation into the ER Initial approaches probing
a stalled translocation intermediate in vitro revealed that
direct cross-links formed only between transiting segments
of translocation substrate and Sec61 (Musch et al 1992
Sanders et al 1992 Mothes et al 1994) Puri1047297cation of
functional Sec61 complex revealed a heterotrimeric complex
consisting of Sec61 associated with two 10-kDa proteins
identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-
cient post-translational translocation the Sec61 complex
assembles with another multimeric membrane complex
termed the Sec63 complex which consists of the genetically
identi1047297ed components Sec63 Sec62 Sec71 and Sec72
(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner
et al 1995) Puri1047297cation of these complexes combined with
proteoliposome reconstitution approaches have demon-
strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein
Kar2 are suf 1047297cient for the post-translational mode of
translocation (Panzner et al 1995) Further biochemical dis-
section of this minimally reconstituted system in addition to
crystal structures of the homologous archaeal SecY complex
(Van den Berg et al 2004) have provided molecular insights
into the translocation mechanism (Rapoport 2007) Current
models for post-translational translocation suggest that the
hydrophobic N-terminal signal sequence is recognized and
bound initially by the Sec63 complex which then transmits
information through conformational changes to the Sec61
complex and to lumenally associated Kar2 (Figure 1b) In
a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an
interface between membrane lipids and speci1047297c transmem-
brane segments in Sec61 where it binds near the cytosolic
face of the channel (Plath et al 1998) Opening of the pore
would then permit a portion of the hydrophilic polypeptide
to span the channel where association with lumenal Kar2
would capture and drive directed movement in a ratcheting
mechanism through cycles of ATP-dependent Kar2 binding
(Neupert et al 1990 Matlack et al 1999) Well-documented
genetic and biochemical interactions between Kar2 and the
lumenal DnaJ domain in Sec63 are thought to coordinate
directed movement into the ER lumen (Feldheim et al
1992 Scidmore et al 1993 Misselwitz et al 1999) The
N-terminal signal sequence is thought to remain bound
at the cytosolic face of the Sec61 complex as the nascent
polypeptide chain is threaded through the pore where at
some stage the signal sequence is cleaved by a translocon-
associated signal peptidase for release into the lumen (Antonin
et al 2000)
Of course a major pathway for delivery of nascent
secretory proteins to the ER employs the signal recognition
particle in a co-translational translocation mechanism Here
the ribosomendashnascent chainndashSRP complex is targeted to
Sec61 translocons through an initial interaction between
SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate
GTP-dependent mechanism paused SRP complexes bound
to SR transfer ribosomendashnascent chains to Sec61 tranlocons
as polypeptide translation continues in a cotranslational
translocation mode (Wild et al 2004) Genetic screens un-
covered the Sec65 subunit of SRP and puri1047297cation of native
SRP identi1047297ed the other core subunits termed Srp14 Srp21
Srp54 Srp68 and Srp72 in addition to the RNA component
encoded by SCR1 (Hann and Walter 1991 Brown et al
Early Events in Protein Secretion 387
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 228
CONTENTS continued
Transport From the ER Sculpting and Populating a COPII Vesicle 391
Structure and assembly of the COPII coat 392
Cargo capture stochastic sampling vs direct and indirect selection 393
Regulation of COPII function GTPase modulation coat modi 1047297 cation 394
Higher-order organization of vesicle formation 395
Vesicle Delivery to the Golgi 395
Vesicle tethering 395
SNARE protein-dependent membrane fusion 396
A concerted model for COPII vesicle tethering and fusion 397
Traf1047297c Within the Golgi 397
Transport through the Golgi complex 397
Lipid requirements for Golgi transport 398
The Return Journey Retrograde Traf1047297c via
COPI Vesicles 398
Composition and structure of the COPI coat 399
Cargo capture sorting signals cargo adaptors and coat stimulators 400
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion 401
Perspectives 401
LIKE all eukaryotes yeast cells segregate various physio-
logical functions into distinct subcellular compartments
A key challenge is thus ensuring that appropriate proteins
are delivered to the correct subcellular destination a process
that is driven by discrete sorting signals that reside in the
proteins themselves Perhaps the most prevalent type of sort-
ing signal is that directing a protein to the secretory pathway
which handles the various proteins that are destined for the
extracellular environment or retention in the internal endo-
membrane system Approximately one-third of the yeast pro-
teome enters the secretory pathway Protein secretion is not
only essential for cellular function but also provides the
driving force for cell growth via delivery of newly synthe-
sized lipid and protein that permits cell expansion Secretory
proteins enter this set of interconnected organelles at the
endoplasmic reticulum (ER) which regulates protein trans-
lation protein translocation across the membrane protein
folding and post-translational modi1047297cation protein quality
control and forward traf 1047297c of suitable cargo molecules (both
lipid and protein) Once contained within the secretory path-
way proteins are ferried between compartments via trans-port vesicles that bud off from one donor compartment to
fuse with a downstream acceptor compartment thereby
mediating directional traf 1047297c of both lipid and protein The
forward-moving or anterograde pathway is balanced by
a reverse or retrograde pathway that returns escaped resi-
dent proteins and maintains the homeostasis of individual
organelles Early yeast screens pioneered the genetic dissec-
tion of the eukaryotic secretory pathway and were rapidly
followed by biochemical approaches that permitted the mo-
lecular dissection of individual processes of protein biogen-
esis and traf 1047297c Here we discuss the methodologies that
have yielded great insight into the conserved processes that
drive protein secretion in all eukaryotes and describe the
fundamental processes that act to ensure ef 1047297cient and ac-
curate protein secretion The reader is also referred to earlier
comprehensive reviews on these topics (Kaiser et al 1997
Lee et al 2004) as we focus our coverage on more recent
advances
Expanding Methodologies From a Parts Listto Mechanisms and Back to More Parts
Classic screens lay the groundwork in vitro reconstitutionde1047297 nes mechanism
There is no doubt that early seminal yeast genetics ap-
proaches laid the foundation upon which our understand-
ing of protein secretion is built From the original Novick
and Schekman screens that identi1047297ed a host of secretion-
defective ( sec) mutants (Novick and Schekman 1979 Novick
et al 1980) to additional more targeted approaches fromthe Schekman (more secs Deshaies and Schekman 1987
Wuestehube et al 1996) Gallwitz ( ypt Gallwitz et al 1983)
Ferro-Novick (bet Newman and Ferro-Novick 1987) Jones
( pep Jones 1977) Stevens ( vps Rothman et al 1989) and
Emr ( vps Bankaitis et al 1986) labs that expanded the rep-
ertoire of mutants with defects in secretory protein and
membrane biosynthesis the 1047297eld has been blessed with an
abundance of reagents that permitted the characterization
of each branch of the secretory pathway (Schekman and
384 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 328
Novick 2004) Many of these processes are essential con-
served and have direct relevance to issues of human health
yet yeast genetics approaches remain at the forefront in
deciphering molecular mechanisms unraveling cellular re-
dundancy and complexity and appreciating the cross-talk
between different branches of the pathway The strength of
yeast as a model system to probe this complexity lies in the
combination of facile genetics and robust biochemistry that
are afforded by this remarkable organism Indeed the 1047297
eldhas a long history of capitalizing on yeast mutants to inform
biochemical reconstitution approaches that in turn inform
new genetic screening approaches
The most pertinent example of the strength of this
approach is the mechanistic description of the COPII coat
proteins that drive vesicle formation from the endoplasmic
reticulum Classic epistasis analyses of the Novick and
Schekman sec mutants (Novick et al 1980) placed the early
sec genes in order within the secretory pathway sec12
sec13 sec16 and sec23 mutants blocked formation of trans-
port vesicles and induced proliferation of the ER whereas
sec17 sec18 and sec22 mutants blocked vesicle fusion and
caused accumulation of vesicles (Novick et al 1981 Kaiserand Schekman 1990) The subsequent development of
in vitro assays relied in part on the use of these mutants in
biochemical complementation assays (Baker et al 1988
Ruohola et al 1988) Recapitulation of ER ndashGolgi traf 1047297c in
permeabilized yeast cells was perturbed in sec23 mutants
but could be restored by incubation with cytosol prepared
from wild-type cells placing Sec23 as a soluble factor re-
quired for transport vesicle formation (Baker et al 1988)
Further re1047297nement of these in vitro transport assays permit-
ted the dissection of different transport stages (Rexach and
Schekman 1991) and allowed the biochemical characteriza-
tion of the COPII coat proteins (Barlowe et al 1994) that
generate transport intermediates and the membrane-bound
and cytosolic factors required for tethering and fusion steps
that consume vesicles at the Golgi membrane (Barlowe
1997 Cao et al 1998) Further mechanistic dissection came
from even more re1047297ned reconstitution systems that permit-
ted the identi1047297cation of the minimal machinery required to
generate COPII vesicles from synthetic liposomes (Matsuoka
et al 1998ab) and de1047297ned the dynamics of individual
events using real-time assays (Antonny et al 2001)
Similar reconstitution of the COPI-mediated GolgindashER
retrograde pathway in yeast lagged somewhat behind in
part due to equivalent biochemical experiments that were
under way in mammalian cells (Balch et al 1984 Waterset al 1991) Furthermore due to rapid perturbation in for-
ward (ER ndashGolgi) traf 1047297c when the retrograde pathway is
blocked for some time there was confusion over the direc-
tionality of COPI-mediated events (Gaynor and Emr 1997)
Despite these dif 1047297culties in vitro reconstitution of COPI-
coated vesicle formation was ultimately achieved (Spang
and Schekman 1998) and has been similarly dissected
in minimal systems using synthetic liposomes (Spang et al
1998)
In contrast to the genetics-informed biochemical ap-
proaches described above minimal reconstitution of the
membrane fusion events that drive vesicle consumption took
a slightly different path Armed with the knowledge that
fusion is driven by proteins known as SNAREs (soluble N-
ethylmaleimide-sensitive factor attachment protein recep-
tors) and with the full description of yeast SNAREs in hand
from computational analyses of the yeast genome Rothman
and colleagues established liposome-based assays that dem-onstrated compartment speci1047297city of different SNARE pairs
(McNew et al 2000) That this biochemical approach largely
recapitulated known pathways previously de1047297ned by ge-
netic means serves to highlight the success of mutually in-
formed genetic and biochemical approaches to fully dissect
the molecular mechanisms of budding and fusion events
Dynamics and organization revealed by live cell imaging
With budding and fusion machineries well described in
minimal systems it became apparent that there were still
pieces of the puzzle missing including the roles of some
essential proteins (eg Sec16 Espenshade et al 1995) that
remained unexplained in terms of functionality Further-more some of the more pressing mechanistic questions
could not be answered by biochemical means For example
the mode of protein and lipid traf 1047297c through the Golgi
remained controversial did COPI vesicles mediate forward
traf 1047297c or did proteins proceed through the Golgi by a process
of maturation of individual cisternae These questions were
addressed in part by the Glick and Nakano labs using high-
resolution time-lapse imaging of living yeast cells (Losev
et al 2006 Matsuura-Tokita et al 2006) Such experiments
de1047297ned discrete sites of vesicle formation known as transi-
tional ER (tER) or ER exit sites (ERES) that are dynamic in
nature can form de novo but also fuse with each other and
have clear relationships with downstream Golgi elements
(Bevis et al 2002 Shindiapina and Barlowe 2010) Further-
more imaging of distinct Golgi elements lent support for the
cisternal maturation model of protein secretion although
direct imaging of cargo molecules remains to be fully dem-
onstrated Recent advances in superresolution imaging hold
great promise in further understanding the nature of these
subdomains and their relationships with distinct protein
machineries and membrane compartments although some
limitations will still apply especially with respect to the
problem of detecting transient cargo molecules that are
in 1047298ux through the system
New technologies yield new players and de1047297 ne interplay between pathways
Since the yeast community entered the postgenomic world
a host of new tools has opened up many new approaches
the haploid deletion collection represents an accessible
large-scale analysis platform for novel screens (Tong
et al 2001) the GFP- (Huh et al 2003) and TAP-tagged
(Ghaemmaghami et al 2003) fusion databases documented
the localization and abundance of many gene products and
Early Events in Protein Secretion 385
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 428
microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation
(Travers et al 2000) These new tools are being used with
remarkable imagination often capitalizing on the facile na-
ture of yeast genetics to de1047297ne the interplay between related
pathways in exciting ways For example microarray analysis
of the changes in gene expression that occur upon induction
of ER stress via the unfolded protein response (UPR) iden-
ti1047297ed upregulation of machineries involved in ER-associated
degradation (ERAD) ultimately leading to the appreciation
that these discrete pathways are intimately coordinated to
manage the burden of protein within the ER (Travers et al
2000) A second example derives from the development of
synthetic genetic array (SGA) technology which allows the
rapid generation of haploid double mutant strains (Tong
et al 2001) Although the piecemeal application of this tech-
nology was informative for individual genes the broader
application to an entire pathway was revolutionary in terms
of being able to de1047297ne novel functions based on shared
genetic 1047297ngerprints The 1047297rst so-called epistatic miniarray
pro1047297le (E-MAP) made pairwise double mutations among
almost 500 early secretory pathway components quantify-
ing the phenotypic cost of combined mutations (Schuldiner
et al 2005) Analysis of the shared patterns of genetic inter-
actions revealed (perhaps not surprisingly) that components
in common pathways shared similar pro1047297les which allowedthe assignation of novel functions to previously uncharacter-
ized and enigmatic proteins An elaboration on the E-MAP
approach made elegant use of a 1047298uorescent reporter system
to 1047297rst assess the UPR state of individual strains in the
genomic deletion collection and then to probe how UPR
activation changes in double mutant backgrounds yielding
a more subtle understanding of genetic interactions than
gross life and death dichotomies which usually form the
basis of synthetic interactions (Jonikas et al 2009) With
further development of such reporters on cell status thisarea of cross-talk between pathways will become more
and more integrated allowing a detailed picture of cellu-
lar physiology However as these new technologies yield
new functional clues to previously uncharacterized genes
we need to continue to use and develop biochemical tools
that allow true mechanistic insight Again the strength of
the yeast system is the use of both genetic and biochemical
tools to mutually inform new discoveries
Secretory Protein Translocation and Biogenesis
Polypeptide targeting and translocation
The 1047297rst step in biogenesis of most secretory proteins is
signal sequence-directed translocation of the polypeptide
into the ER Both cotranslational and post-translational
mechanisms operate in yeast to target diverse sets of soluble
and integral membrane secretory proteins to the ER (Figure
1) The cotranslational translocation process is initiated
when a hydrophobic signal sequence or transmembrane
sequence is translated and recognized by the signal-recognition
particle (SRP) for targeting to the SRP receptor at ER trans-
location sites (Figure 1a) In the case of post-translational
translocation cytosolic chaperones play a critical role in
binding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-
location competent state until delivery to the ER membrane
(Figure 1b) Progress on identi1047297cation and characterization
of the translocation machinery will be described in turn
below as the start of a continuum of events in biogenesis
of secretory proteins
Genetic approaches in yeast uncovered key components
in both the co- and post-translational translocation path-
ways Appending a signal sequence to the cytosolic enzyme
Figure 1 Membrane transloca-
tion of secretory proteins Three
well-characterized pathways oper-
ate to deliver secretory proteins
to the ER for membrane trans-
location (A) The signal recogni-
tion particle (SRP) recognizes a
hydrophobic signal sequence or
transmembrane segment during
protein translation followed by
targeting of the ribosomendash
nascentchain complex to the SRP receptor
for cotranslational membrane in-
sertion (B) Post-translational inser-
tion of secretory proteins depends
on cytosolic Hsp70 ATPases such
as Ssa1 to maintain the nascent
secretory protein in an unfolded
translocation competent state until delivery to the Sec63 complex formed by Sec62Sec63Sec71Sec72 The Sec61 complex forms an aqueous
channel for both post- and cotranslational polypeptide translocation Kar2 a luminal Hsp70 ATPase facilitates directed movement and folding
of nascent polypeptides (C) In GET-mediated insertion of C-terminal tail-anchored proteins the Sgt2ndashGet4ndashGet5 complex targets nascent
polypeptides to Get3 for Get1Get2 dependent translocation Tail-anchored proteins are integrated into the membrane in Sec61-independent
pathway
386 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 528
encoded by HIS4 targets this enzyme to the ER where it
cannot function and produces histidine auxotrophy A ge-
netic selection for mutants that are partially defective in
translocation of this signal peptide-bearing fusion protein
and therefore restore histidine prototrophy was used to
identify conditional mutations in three essential genes
SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987
Rothblatt et al 1989) Sequencing indicated that all three
genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-
membrane segments and striking sequence identity with the
Escherichia coli translocation protein SecY (Stirling et al
1992 Jungnickel et al 1994) Similar genetic selection
approaches using the HIS4 gene product fused to integral
membrane proteins identi1047297ed SEC65 which encodes a com-
ponent of the SRP (Stirling and Hewitt 1992 Stirling et al
1992) as well as mutations in SEC71 and SEC72 (Green
et al 1992)
Concurrent with these genetic approaches cell-free
reconstitution assays that measured post-translational
translocation of radiolabeled pre-pro-a-factor into yeast
microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt
and Meyer 1986) Fractionation of cytosolic components re-
quired in the cell-free assay revealed that Hsp70 ATPases
stimulated post-translational translocation (Chirico et al
1988) Yeast express a partially redundant family of cyto-
solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-
lectively essential An in vivo test for Hsp70 function in
translocation was demonstrated when conditional expres-
sion of SSA1 in the background of the multiple ssa D strain
resulted in accumulation of unprocessed secretory proteins
as Ssa1 was depleted (Deshaies et al 1988) ATPase activity
of Hsp70 family members is often stimulated by a corre-
sponding Hsp40 Dna J partner and in the case of poly-
peptide translocation in yeast the YDJ1 gene encodes
a farnsylated DnaJ homolog that functions in ER transloca-
tion (Caplan et al 1992) Ydj1 has been shown to directly
regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer
et al 1995) and structural studies indicate that Ydj1 binds to
three- to four-residue hydrophobic stretches in nonnative
proteins that are then presented to Hsp70 proteins such as
Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-
ments connect YDJ1 to translocation components in addi-
tion to multiple other cellular pathways presumably due to
action on a subset of secretory proteins (Becker et al 1996
Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the
multispanning Sec61 forms an aqueous channel for polypep-
tide translocation into the ER Initial approaches probing
a stalled translocation intermediate in vitro revealed that
direct cross-links formed only between transiting segments
of translocation substrate and Sec61 (Musch et al 1992
Sanders et al 1992 Mothes et al 1994) Puri1047297cation of
functional Sec61 complex revealed a heterotrimeric complex
consisting of Sec61 associated with two 10-kDa proteins
identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-
cient post-translational translocation the Sec61 complex
assembles with another multimeric membrane complex
termed the Sec63 complex which consists of the genetically
identi1047297ed components Sec63 Sec62 Sec71 and Sec72
(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner
et al 1995) Puri1047297cation of these complexes combined with
proteoliposome reconstitution approaches have demon-
strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein
Kar2 are suf 1047297cient for the post-translational mode of
translocation (Panzner et al 1995) Further biochemical dis-
section of this minimally reconstituted system in addition to
crystal structures of the homologous archaeal SecY complex
(Van den Berg et al 2004) have provided molecular insights
into the translocation mechanism (Rapoport 2007) Current
models for post-translational translocation suggest that the
hydrophobic N-terminal signal sequence is recognized and
bound initially by the Sec63 complex which then transmits
information through conformational changes to the Sec61
complex and to lumenally associated Kar2 (Figure 1b) In
a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an
interface between membrane lipids and speci1047297c transmem-
brane segments in Sec61 where it binds near the cytosolic
face of the channel (Plath et al 1998) Opening of the pore
would then permit a portion of the hydrophilic polypeptide
to span the channel where association with lumenal Kar2
would capture and drive directed movement in a ratcheting
mechanism through cycles of ATP-dependent Kar2 binding
(Neupert et al 1990 Matlack et al 1999) Well-documented
genetic and biochemical interactions between Kar2 and the
lumenal DnaJ domain in Sec63 are thought to coordinate
directed movement into the ER lumen (Feldheim et al
1992 Scidmore et al 1993 Misselwitz et al 1999) The
N-terminal signal sequence is thought to remain bound
at the cytosolic face of the Sec61 complex as the nascent
polypeptide chain is threaded through the pore where at
some stage the signal sequence is cleaved by a translocon-
associated signal peptidase for release into the lumen (Antonin
et al 2000)
Of course a major pathway for delivery of nascent
secretory proteins to the ER employs the signal recognition
particle in a co-translational translocation mechanism Here
the ribosomendashnascent chainndashSRP complex is targeted to
Sec61 translocons through an initial interaction between
SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate
GTP-dependent mechanism paused SRP complexes bound
to SR transfer ribosomendashnascent chains to Sec61 tranlocons
as polypeptide translation continues in a cotranslational
translocation mode (Wild et al 2004) Genetic screens un-
covered the Sec65 subunit of SRP and puri1047297cation of native
SRP identi1047297ed the other core subunits termed Srp14 Srp21
Srp54 Srp68 and Srp72 in addition to the RNA component
encoded by SCR1 (Hann and Walter 1991 Brown et al
Early Events in Protein Secretion 387
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 328
Novick 2004) Many of these processes are essential con-
served and have direct relevance to issues of human health
yet yeast genetics approaches remain at the forefront in
deciphering molecular mechanisms unraveling cellular re-
dundancy and complexity and appreciating the cross-talk
between different branches of the pathway The strength of
yeast as a model system to probe this complexity lies in the
combination of facile genetics and robust biochemistry that
are afforded by this remarkable organism Indeed the 1047297
eldhas a long history of capitalizing on yeast mutants to inform
biochemical reconstitution approaches that in turn inform
new genetic screening approaches
The most pertinent example of the strength of this
approach is the mechanistic description of the COPII coat
proteins that drive vesicle formation from the endoplasmic
reticulum Classic epistasis analyses of the Novick and
Schekman sec mutants (Novick et al 1980) placed the early
sec genes in order within the secretory pathway sec12
sec13 sec16 and sec23 mutants blocked formation of trans-
port vesicles and induced proliferation of the ER whereas
sec17 sec18 and sec22 mutants blocked vesicle fusion and
caused accumulation of vesicles (Novick et al 1981 Kaiserand Schekman 1990) The subsequent development of
in vitro assays relied in part on the use of these mutants in
biochemical complementation assays (Baker et al 1988
Ruohola et al 1988) Recapitulation of ER ndashGolgi traf 1047297c in
permeabilized yeast cells was perturbed in sec23 mutants
but could be restored by incubation with cytosol prepared
from wild-type cells placing Sec23 as a soluble factor re-
quired for transport vesicle formation (Baker et al 1988)
Further re1047297nement of these in vitro transport assays permit-
ted the dissection of different transport stages (Rexach and
Schekman 1991) and allowed the biochemical characteriza-
tion of the COPII coat proteins (Barlowe et al 1994) that
generate transport intermediates and the membrane-bound
and cytosolic factors required for tethering and fusion steps
that consume vesicles at the Golgi membrane (Barlowe
1997 Cao et al 1998) Further mechanistic dissection came
from even more re1047297ned reconstitution systems that permit-
ted the identi1047297cation of the minimal machinery required to
generate COPII vesicles from synthetic liposomes (Matsuoka
et al 1998ab) and de1047297ned the dynamics of individual
events using real-time assays (Antonny et al 2001)
Similar reconstitution of the COPI-mediated GolgindashER
retrograde pathway in yeast lagged somewhat behind in
part due to equivalent biochemical experiments that were
under way in mammalian cells (Balch et al 1984 Waterset al 1991) Furthermore due to rapid perturbation in for-
ward (ER ndashGolgi) traf 1047297c when the retrograde pathway is
blocked for some time there was confusion over the direc-
tionality of COPI-mediated events (Gaynor and Emr 1997)
Despite these dif 1047297culties in vitro reconstitution of COPI-
coated vesicle formation was ultimately achieved (Spang
and Schekman 1998) and has been similarly dissected
in minimal systems using synthetic liposomes (Spang et al
1998)
In contrast to the genetics-informed biochemical ap-
proaches described above minimal reconstitution of the
membrane fusion events that drive vesicle consumption took
a slightly different path Armed with the knowledge that
fusion is driven by proteins known as SNAREs (soluble N-
ethylmaleimide-sensitive factor attachment protein recep-
tors) and with the full description of yeast SNAREs in hand
from computational analyses of the yeast genome Rothman
and colleagues established liposome-based assays that dem-onstrated compartment speci1047297city of different SNARE pairs
(McNew et al 2000) That this biochemical approach largely
recapitulated known pathways previously de1047297ned by ge-
netic means serves to highlight the success of mutually in-
formed genetic and biochemical approaches to fully dissect
the molecular mechanisms of budding and fusion events
Dynamics and organization revealed by live cell imaging
With budding and fusion machineries well described in
minimal systems it became apparent that there were still
pieces of the puzzle missing including the roles of some
essential proteins (eg Sec16 Espenshade et al 1995) that
remained unexplained in terms of functionality Further-more some of the more pressing mechanistic questions
could not be answered by biochemical means For example
the mode of protein and lipid traf 1047297c through the Golgi
remained controversial did COPI vesicles mediate forward
traf 1047297c or did proteins proceed through the Golgi by a process
of maturation of individual cisternae These questions were
addressed in part by the Glick and Nakano labs using high-
resolution time-lapse imaging of living yeast cells (Losev
et al 2006 Matsuura-Tokita et al 2006) Such experiments
de1047297ned discrete sites of vesicle formation known as transi-
tional ER (tER) or ER exit sites (ERES) that are dynamic in
nature can form de novo but also fuse with each other and
have clear relationships with downstream Golgi elements
(Bevis et al 2002 Shindiapina and Barlowe 2010) Further-
more imaging of distinct Golgi elements lent support for the
cisternal maturation model of protein secretion although
direct imaging of cargo molecules remains to be fully dem-
onstrated Recent advances in superresolution imaging hold
great promise in further understanding the nature of these
subdomains and their relationships with distinct protein
machineries and membrane compartments although some
limitations will still apply especially with respect to the
problem of detecting transient cargo molecules that are
in 1047298ux through the system
New technologies yield new players and de1047297 ne interplay between pathways
Since the yeast community entered the postgenomic world
a host of new tools has opened up many new approaches
the haploid deletion collection represents an accessible
large-scale analysis platform for novel screens (Tong
et al 2001) the GFP- (Huh et al 2003) and TAP-tagged
(Ghaemmaghami et al 2003) fusion databases documented
the localization and abundance of many gene products and
Early Events in Protein Secretion 385
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 428
microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation
(Travers et al 2000) These new tools are being used with
remarkable imagination often capitalizing on the facile na-
ture of yeast genetics to de1047297ne the interplay between related
pathways in exciting ways For example microarray analysis
of the changes in gene expression that occur upon induction
of ER stress via the unfolded protein response (UPR) iden-
ti1047297ed upregulation of machineries involved in ER-associated
degradation (ERAD) ultimately leading to the appreciation
that these discrete pathways are intimately coordinated to
manage the burden of protein within the ER (Travers et al
2000) A second example derives from the development of
synthetic genetic array (SGA) technology which allows the
rapid generation of haploid double mutant strains (Tong
et al 2001) Although the piecemeal application of this tech-
nology was informative for individual genes the broader
application to an entire pathway was revolutionary in terms
of being able to de1047297ne novel functions based on shared
genetic 1047297ngerprints The 1047297rst so-called epistatic miniarray
pro1047297le (E-MAP) made pairwise double mutations among
almost 500 early secretory pathway components quantify-
ing the phenotypic cost of combined mutations (Schuldiner
et al 2005) Analysis of the shared patterns of genetic inter-
actions revealed (perhaps not surprisingly) that components
in common pathways shared similar pro1047297les which allowedthe assignation of novel functions to previously uncharacter-
ized and enigmatic proteins An elaboration on the E-MAP
approach made elegant use of a 1047298uorescent reporter system
to 1047297rst assess the UPR state of individual strains in the
genomic deletion collection and then to probe how UPR
activation changes in double mutant backgrounds yielding
a more subtle understanding of genetic interactions than
gross life and death dichotomies which usually form the
basis of synthetic interactions (Jonikas et al 2009) With
further development of such reporters on cell status thisarea of cross-talk between pathways will become more
and more integrated allowing a detailed picture of cellu-
lar physiology However as these new technologies yield
new functional clues to previously uncharacterized genes
we need to continue to use and develop biochemical tools
that allow true mechanistic insight Again the strength of
the yeast system is the use of both genetic and biochemical
tools to mutually inform new discoveries
Secretory Protein Translocation and Biogenesis
Polypeptide targeting and translocation
The 1047297rst step in biogenesis of most secretory proteins is
signal sequence-directed translocation of the polypeptide
into the ER Both cotranslational and post-translational
mechanisms operate in yeast to target diverse sets of soluble
and integral membrane secretory proteins to the ER (Figure
1) The cotranslational translocation process is initiated
when a hydrophobic signal sequence or transmembrane
sequence is translated and recognized by the signal-recognition
particle (SRP) for targeting to the SRP receptor at ER trans-
location sites (Figure 1a) In the case of post-translational
translocation cytosolic chaperones play a critical role in
binding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-
location competent state until delivery to the ER membrane
(Figure 1b) Progress on identi1047297cation and characterization
of the translocation machinery will be described in turn
below as the start of a continuum of events in biogenesis
of secretory proteins
Genetic approaches in yeast uncovered key components
in both the co- and post-translational translocation path-
ways Appending a signal sequence to the cytosolic enzyme
Figure 1 Membrane transloca-
tion of secretory proteins Three
well-characterized pathways oper-
ate to deliver secretory proteins
to the ER for membrane trans-
location (A) The signal recogni-
tion particle (SRP) recognizes a
hydrophobic signal sequence or
transmembrane segment during
protein translation followed by
targeting of the ribosomendash
nascentchain complex to the SRP receptor
for cotranslational membrane in-
sertion (B) Post-translational inser-
tion of secretory proteins depends
on cytosolic Hsp70 ATPases such
as Ssa1 to maintain the nascent
secretory protein in an unfolded
translocation competent state until delivery to the Sec63 complex formed by Sec62Sec63Sec71Sec72 The Sec61 complex forms an aqueous
channel for both post- and cotranslational polypeptide translocation Kar2 a luminal Hsp70 ATPase facilitates directed movement and folding
of nascent polypeptides (C) In GET-mediated insertion of C-terminal tail-anchored proteins the Sgt2ndashGet4ndashGet5 complex targets nascent
polypeptides to Get3 for Get1Get2 dependent translocation Tail-anchored proteins are integrated into the membrane in Sec61-independent
pathway
386 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 528
encoded by HIS4 targets this enzyme to the ER where it
cannot function and produces histidine auxotrophy A ge-
netic selection for mutants that are partially defective in
translocation of this signal peptide-bearing fusion protein
and therefore restore histidine prototrophy was used to
identify conditional mutations in three essential genes
SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987
Rothblatt et al 1989) Sequencing indicated that all three
genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-
membrane segments and striking sequence identity with the
Escherichia coli translocation protein SecY (Stirling et al
1992 Jungnickel et al 1994) Similar genetic selection
approaches using the HIS4 gene product fused to integral
membrane proteins identi1047297ed SEC65 which encodes a com-
ponent of the SRP (Stirling and Hewitt 1992 Stirling et al
1992) as well as mutations in SEC71 and SEC72 (Green
et al 1992)
Concurrent with these genetic approaches cell-free
reconstitution assays that measured post-translational
translocation of radiolabeled pre-pro-a-factor into yeast
microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt
and Meyer 1986) Fractionation of cytosolic components re-
quired in the cell-free assay revealed that Hsp70 ATPases
stimulated post-translational translocation (Chirico et al
1988) Yeast express a partially redundant family of cyto-
solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-
lectively essential An in vivo test for Hsp70 function in
translocation was demonstrated when conditional expres-
sion of SSA1 in the background of the multiple ssa D strain
resulted in accumulation of unprocessed secretory proteins
as Ssa1 was depleted (Deshaies et al 1988) ATPase activity
of Hsp70 family members is often stimulated by a corre-
sponding Hsp40 Dna J partner and in the case of poly-
peptide translocation in yeast the YDJ1 gene encodes
a farnsylated DnaJ homolog that functions in ER transloca-
tion (Caplan et al 1992) Ydj1 has been shown to directly
regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer
et al 1995) and structural studies indicate that Ydj1 binds to
three- to four-residue hydrophobic stretches in nonnative
proteins that are then presented to Hsp70 proteins such as
Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-
ments connect YDJ1 to translocation components in addi-
tion to multiple other cellular pathways presumably due to
action on a subset of secretory proteins (Becker et al 1996
Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the
multispanning Sec61 forms an aqueous channel for polypep-
tide translocation into the ER Initial approaches probing
a stalled translocation intermediate in vitro revealed that
direct cross-links formed only between transiting segments
of translocation substrate and Sec61 (Musch et al 1992
Sanders et al 1992 Mothes et al 1994) Puri1047297cation of
functional Sec61 complex revealed a heterotrimeric complex
consisting of Sec61 associated with two 10-kDa proteins
identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-
cient post-translational translocation the Sec61 complex
assembles with another multimeric membrane complex
termed the Sec63 complex which consists of the genetically
identi1047297ed components Sec63 Sec62 Sec71 and Sec72
(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner
et al 1995) Puri1047297cation of these complexes combined with
proteoliposome reconstitution approaches have demon-
strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein
Kar2 are suf 1047297cient for the post-translational mode of
translocation (Panzner et al 1995) Further biochemical dis-
section of this minimally reconstituted system in addition to
crystal structures of the homologous archaeal SecY complex
(Van den Berg et al 2004) have provided molecular insights
into the translocation mechanism (Rapoport 2007) Current
models for post-translational translocation suggest that the
hydrophobic N-terminal signal sequence is recognized and
bound initially by the Sec63 complex which then transmits
information through conformational changes to the Sec61
complex and to lumenally associated Kar2 (Figure 1b) In
a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an
interface between membrane lipids and speci1047297c transmem-
brane segments in Sec61 where it binds near the cytosolic
face of the channel (Plath et al 1998) Opening of the pore
would then permit a portion of the hydrophilic polypeptide
to span the channel where association with lumenal Kar2
would capture and drive directed movement in a ratcheting
mechanism through cycles of ATP-dependent Kar2 binding
(Neupert et al 1990 Matlack et al 1999) Well-documented
genetic and biochemical interactions between Kar2 and the
lumenal DnaJ domain in Sec63 are thought to coordinate
directed movement into the ER lumen (Feldheim et al
1992 Scidmore et al 1993 Misselwitz et al 1999) The
N-terminal signal sequence is thought to remain bound
at the cytosolic face of the Sec61 complex as the nascent
polypeptide chain is threaded through the pore where at
some stage the signal sequence is cleaved by a translocon-
associated signal peptidase for release into the lumen (Antonin
et al 2000)
Of course a major pathway for delivery of nascent
secretory proteins to the ER employs the signal recognition
particle in a co-translational translocation mechanism Here
the ribosomendashnascent chainndashSRP complex is targeted to
Sec61 translocons through an initial interaction between
SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate
GTP-dependent mechanism paused SRP complexes bound
to SR transfer ribosomendashnascent chains to Sec61 tranlocons
as polypeptide translation continues in a cotranslational
translocation mode (Wild et al 2004) Genetic screens un-
covered the Sec65 subunit of SRP and puri1047297cation of native
SRP identi1047297ed the other core subunits termed Srp14 Srp21
Srp54 Srp68 and Srp72 in addition to the RNA component
encoded by SCR1 (Hann and Walter 1991 Brown et al
Early Events in Protein Secretion 387
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 428
microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation
(Travers et al 2000) These new tools are being used with
remarkable imagination often capitalizing on the facile na-
ture of yeast genetics to de1047297ne the interplay between related
pathways in exciting ways For example microarray analysis
of the changes in gene expression that occur upon induction
of ER stress via the unfolded protein response (UPR) iden-
ti1047297ed upregulation of machineries involved in ER-associated
degradation (ERAD) ultimately leading to the appreciation
that these discrete pathways are intimately coordinated to
manage the burden of protein within the ER (Travers et al
2000) A second example derives from the development of
synthetic genetic array (SGA) technology which allows the
rapid generation of haploid double mutant strains (Tong
et al 2001) Although the piecemeal application of this tech-
nology was informative for individual genes the broader
application to an entire pathway was revolutionary in terms
of being able to de1047297ne novel functions based on shared
genetic 1047297ngerprints The 1047297rst so-called epistatic miniarray
pro1047297le (E-MAP) made pairwise double mutations among
almost 500 early secretory pathway components quantify-
ing the phenotypic cost of combined mutations (Schuldiner
et al 2005) Analysis of the shared patterns of genetic inter-
actions revealed (perhaps not surprisingly) that components
in common pathways shared similar pro1047297les which allowedthe assignation of novel functions to previously uncharacter-
ized and enigmatic proteins An elaboration on the E-MAP
approach made elegant use of a 1047298uorescent reporter system
to 1047297rst assess the UPR state of individual strains in the
genomic deletion collection and then to probe how UPR
activation changes in double mutant backgrounds yielding
a more subtle understanding of genetic interactions than
gross life and death dichotomies which usually form the
basis of synthetic interactions (Jonikas et al 2009) With
further development of such reporters on cell status thisarea of cross-talk between pathways will become more
and more integrated allowing a detailed picture of cellu-
lar physiology However as these new technologies yield
new functional clues to previously uncharacterized genes
we need to continue to use and develop biochemical tools
that allow true mechanistic insight Again the strength of
the yeast system is the use of both genetic and biochemical
tools to mutually inform new discoveries
Secretory Protein Translocation and Biogenesis
Polypeptide targeting and translocation
The 1047297rst step in biogenesis of most secretory proteins is
signal sequence-directed translocation of the polypeptide
into the ER Both cotranslational and post-translational
mechanisms operate in yeast to target diverse sets of soluble
and integral membrane secretory proteins to the ER (Figure
1) The cotranslational translocation process is initiated
when a hydrophobic signal sequence or transmembrane
sequence is translated and recognized by the signal-recognition
particle (SRP) for targeting to the SRP receptor at ER trans-
location sites (Figure 1a) In the case of post-translational
translocation cytosolic chaperones play a critical role in
binding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-
location competent state until delivery to the ER membrane
(Figure 1b) Progress on identi1047297cation and characterization
of the translocation machinery will be described in turn
below as the start of a continuum of events in biogenesis
of secretory proteins
Genetic approaches in yeast uncovered key components
in both the co- and post-translational translocation path-
ways Appending a signal sequence to the cytosolic enzyme
Figure 1 Membrane transloca-
tion of secretory proteins Three
well-characterized pathways oper-
ate to deliver secretory proteins
to the ER for membrane trans-
location (A) The signal recogni-
tion particle (SRP) recognizes a
hydrophobic signal sequence or
transmembrane segment during
protein translation followed by
targeting of the ribosomendash
nascentchain complex to the SRP receptor
for cotranslational membrane in-
sertion (B) Post-translational inser-
tion of secretory proteins depends
on cytosolic Hsp70 ATPases such
as Ssa1 to maintain the nascent
secretory protein in an unfolded
translocation competent state until delivery to the Sec63 complex formed by Sec62Sec63Sec71Sec72 The Sec61 complex forms an aqueous
channel for both post- and cotranslational polypeptide translocation Kar2 a luminal Hsp70 ATPase facilitates directed movement and folding
of nascent polypeptides (C) In GET-mediated insertion of C-terminal tail-anchored proteins the Sgt2ndashGet4ndashGet5 complex targets nascent
polypeptides to Get3 for Get1Get2 dependent translocation Tail-anchored proteins are integrated into the membrane in Sec61-independent
pathway
386 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 528
encoded by HIS4 targets this enzyme to the ER where it
cannot function and produces histidine auxotrophy A ge-
netic selection for mutants that are partially defective in
translocation of this signal peptide-bearing fusion protein
and therefore restore histidine prototrophy was used to
identify conditional mutations in three essential genes
SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987
Rothblatt et al 1989) Sequencing indicated that all three
genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-
membrane segments and striking sequence identity with the
Escherichia coli translocation protein SecY (Stirling et al
1992 Jungnickel et al 1994) Similar genetic selection
approaches using the HIS4 gene product fused to integral
membrane proteins identi1047297ed SEC65 which encodes a com-
ponent of the SRP (Stirling and Hewitt 1992 Stirling et al
1992) as well as mutations in SEC71 and SEC72 (Green
et al 1992)
Concurrent with these genetic approaches cell-free
reconstitution assays that measured post-translational
translocation of radiolabeled pre-pro-a-factor into yeast
microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt
and Meyer 1986) Fractionation of cytosolic components re-
quired in the cell-free assay revealed that Hsp70 ATPases
stimulated post-translational translocation (Chirico et al
1988) Yeast express a partially redundant family of cyto-
solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-
lectively essential An in vivo test for Hsp70 function in
translocation was demonstrated when conditional expres-
sion of SSA1 in the background of the multiple ssa D strain
resulted in accumulation of unprocessed secretory proteins
as Ssa1 was depleted (Deshaies et al 1988) ATPase activity
of Hsp70 family members is often stimulated by a corre-
sponding Hsp40 Dna J partner and in the case of poly-
peptide translocation in yeast the YDJ1 gene encodes
a farnsylated DnaJ homolog that functions in ER transloca-
tion (Caplan et al 1992) Ydj1 has been shown to directly
regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer
et al 1995) and structural studies indicate that Ydj1 binds to
three- to four-residue hydrophobic stretches in nonnative
proteins that are then presented to Hsp70 proteins such as
Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-
ments connect YDJ1 to translocation components in addi-
tion to multiple other cellular pathways presumably due to
action on a subset of secretory proteins (Becker et al 1996
Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the
multispanning Sec61 forms an aqueous channel for polypep-
tide translocation into the ER Initial approaches probing
a stalled translocation intermediate in vitro revealed that
direct cross-links formed only between transiting segments
of translocation substrate and Sec61 (Musch et al 1992
Sanders et al 1992 Mothes et al 1994) Puri1047297cation of
functional Sec61 complex revealed a heterotrimeric complex
consisting of Sec61 associated with two 10-kDa proteins
identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-
cient post-translational translocation the Sec61 complex
assembles with another multimeric membrane complex
termed the Sec63 complex which consists of the genetically
identi1047297ed components Sec63 Sec62 Sec71 and Sec72
(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner
et al 1995) Puri1047297cation of these complexes combined with
proteoliposome reconstitution approaches have demon-
strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein
Kar2 are suf 1047297cient for the post-translational mode of
translocation (Panzner et al 1995) Further biochemical dis-
section of this minimally reconstituted system in addition to
crystal structures of the homologous archaeal SecY complex
(Van den Berg et al 2004) have provided molecular insights
into the translocation mechanism (Rapoport 2007) Current
models for post-translational translocation suggest that the
hydrophobic N-terminal signal sequence is recognized and
bound initially by the Sec63 complex which then transmits
information through conformational changes to the Sec61
complex and to lumenally associated Kar2 (Figure 1b) In
a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an
interface between membrane lipids and speci1047297c transmem-
brane segments in Sec61 where it binds near the cytosolic
face of the channel (Plath et al 1998) Opening of the pore
would then permit a portion of the hydrophilic polypeptide
to span the channel where association with lumenal Kar2
would capture and drive directed movement in a ratcheting
mechanism through cycles of ATP-dependent Kar2 binding
(Neupert et al 1990 Matlack et al 1999) Well-documented
genetic and biochemical interactions between Kar2 and the
lumenal DnaJ domain in Sec63 are thought to coordinate
directed movement into the ER lumen (Feldheim et al
1992 Scidmore et al 1993 Misselwitz et al 1999) The
N-terminal signal sequence is thought to remain bound
at the cytosolic face of the Sec61 complex as the nascent
polypeptide chain is threaded through the pore where at
some stage the signal sequence is cleaved by a translocon-
associated signal peptidase for release into the lumen (Antonin
et al 2000)
Of course a major pathway for delivery of nascent
secretory proteins to the ER employs the signal recognition
particle in a co-translational translocation mechanism Here
the ribosomendashnascent chainndashSRP complex is targeted to
Sec61 translocons through an initial interaction between
SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate
GTP-dependent mechanism paused SRP complexes bound
to SR transfer ribosomendashnascent chains to Sec61 tranlocons
as polypeptide translation continues in a cotranslational
translocation mode (Wild et al 2004) Genetic screens un-
covered the Sec65 subunit of SRP and puri1047297cation of native
SRP identi1047297ed the other core subunits termed Srp14 Srp21
Srp54 Srp68 and Srp72 in addition to the RNA component
encoded by SCR1 (Hann and Walter 1991 Brown et al
Early Events in Protein Secretion 387
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 528
encoded by HIS4 targets this enzyme to the ER where it
cannot function and produces histidine auxotrophy A ge-
netic selection for mutants that are partially defective in
translocation of this signal peptide-bearing fusion protein
and therefore restore histidine prototrophy was used to
identify conditional mutations in three essential genes
SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987
Rothblatt et al 1989) Sequencing indicated that all three
genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-
membrane segments and striking sequence identity with the
Escherichia coli translocation protein SecY (Stirling et al
1992 Jungnickel et al 1994) Similar genetic selection
approaches using the HIS4 gene product fused to integral
membrane proteins identi1047297ed SEC65 which encodes a com-
ponent of the SRP (Stirling and Hewitt 1992 Stirling et al
1992) as well as mutations in SEC71 and SEC72 (Green
et al 1992)
Concurrent with these genetic approaches cell-free
reconstitution assays that measured post-translational
translocation of radiolabeled pre-pro-a-factor into yeast
microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt
and Meyer 1986) Fractionation of cytosolic components re-
quired in the cell-free assay revealed that Hsp70 ATPases
stimulated post-translational translocation (Chirico et al
1988) Yeast express a partially redundant family of cyto-
solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-
lectively essential An in vivo test for Hsp70 function in
translocation was demonstrated when conditional expres-
sion of SSA1 in the background of the multiple ssa D strain
resulted in accumulation of unprocessed secretory proteins
as Ssa1 was depleted (Deshaies et al 1988) ATPase activity
of Hsp70 family members is often stimulated by a corre-
sponding Hsp40 Dna J partner and in the case of poly-
peptide translocation in yeast the YDJ1 gene encodes
a farnsylated DnaJ homolog that functions in ER transloca-
tion (Caplan et al 1992) Ydj1 has been shown to directly
regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer
et al 1995) and structural studies indicate that Ydj1 binds to
three- to four-residue hydrophobic stretches in nonnative
proteins that are then presented to Hsp70 proteins such as
Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-
ments connect YDJ1 to translocation components in addi-
tion to multiple other cellular pathways presumably due to
action on a subset of secretory proteins (Becker et al 1996
Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the
multispanning Sec61 forms an aqueous channel for polypep-
tide translocation into the ER Initial approaches probing
a stalled translocation intermediate in vitro revealed that
direct cross-links formed only between transiting segments
of translocation substrate and Sec61 (Musch et al 1992
Sanders et al 1992 Mothes et al 1994) Puri1047297cation of
functional Sec61 complex revealed a heterotrimeric complex
consisting of Sec61 associated with two 10-kDa proteins
identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-
cient post-translational translocation the Sec61 complex
assembles with another multimeric membrane complex
termed the Sec63 complex which consists of the genetically
identi1047297ed components Sec63 Sec62 Sec71 and Sec72
(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner
et al 1995) Puri1047297cation of these complexes combined with
proteoliposome reconstitution approaches have demon-
strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein
Kar2 are suf 1047297cient for the post-translational mode of
translocation (Panzner et al 1995) Further biochemical dis-
section of this minimally reconstituted system in addition to
crystal structures of the homologous archaeal SecY complex
(Van den Berg et al 2004) have provided molecular insights
into the translocation mechanism (Rapoport 2007) Current
models for post-translational translocation suggest that the
hydrophobic N-terminal signal sequence is recognized and
bound initially by the Sec63 complex which then transmits
information through conformational changes to the Sec61
complex and to lumenally associated Kar2 (Figure 1b) In
a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an
interface between membrane lipids and speci1047297c transmem-
brane segments in Sec61 where it binds near the cytosolic
face of the channel (Plath et al 1998) Opening of the pore
would then permit a portion of the hydrophilic polypeptide
to span the channel where association with lumenal Kar2
would capture and drive directed movement in a ratcheting
mechanism through cycles of ATP-dependent Kar2 binding
(Neupert et al 1990 Matlack et al 1999) Well-documented
genetic and biochemical interactions between Kar2 and the
lumenal DnaJ domain in Sec63 are thought to coordinate
directed movement into the ER lumen (Feldheim et al
1992 Scidmore et al 1993 Misselwitz et al 1999) The
N-terminal signal sequence is thought to remain bound
at the cytosolic face of the Sec61 complex as the nascent
polypeptide chain is threaded through the pore where at
some stage the signal sequence is cleaved by a translocon-
associated signal peptidase for release into the lumen (Antonin
et al 2000)
Of course a major pathway for delivery of nascent
secretory proteins to the ER employs the signal recognition
particle in a co-translational translocation mechanism Here
the ribosomendashnascent chainndashSRP complex is targeted to
Sec61 translocons through an initial interaction between
SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate
GTP-dependent mechanism paused SRP complexes bound
to SR transfer ribosomendashnascent chains to Sec61 tranlocons
as polypeptide translation continues in a cotranslational
translocation mode (Wild et al 2004) Genetic screens un-
covered the Sec65 subunit of SRP and puri1047297cation of native
SRP identi1047297ed the other core subunits termed Srp14 Srp21
Srp54 Srp68 and Srp72 in addition to the RNA component
encoded by SCR1 (Hann and Walter 1991 Brown et al
Early Events in Protein Secretion 387
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 628
1994) Somewhat surprisingly deletion of the SRP compo-
nents in yeast produced yeast cells that grow slowly but
remain viable These 1047297ndings indicate that the SRP-dependent
pathway is not essential unlike the core translocation pore
components and indicates that other cytosolic machinery
can manage delivery of all essential secretory proteins to
the translocon Although yeast cells can tolerate complete
loss of the SRP pathway it became clear that certain secre-
tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the
ER in a post-translational mode (Hann et al 1992 Stirling
and Hewitt 1992) In general integral membrane proteins
and signal sequences of relatively high hydrophobicity pref-
erentially engage the SRP-dependent pathway whereas sol-
uble and lower hydrophobicity signal sequences depend on
a Sec63-mediated post-translational mode of translocation
(Ng et al 1996)
More recently a third post-translational translocation
pathway to the ER has been characterized in yeast and
other eukaryotes whereby short integral membrane proteins
and C-terminal tail-anchored proteins are integrated into
the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-
brane segments are occluded by the ribosome until trans-
lation is completed thereby precluding SRP-dependent
targeting Bioinformatic analyses suggest that up to 5
of predicted integral membrane proteins in eukaryotic
genomes may follow this SRP-independent route including
the large class of SNARE proteins that drive intracellular
membrane fusion events and are anchored by C-terminal
membrane domains Interestingly this post-translational tar-
geting pathway operates independently of the Sec61 and
Sec63 translocon complexes (Steel et al 2002 Yabal et al
2003) and instead depends on recently de1047297ned soluble and
membrane-bound factors Large-scale genetic interaction
analyses in yeast identi1047297ed a clustered set of nonessential
genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that
were named GET genes (Schuldiner et al 2005) Get3
shares high sequence identity with the transmembrane do-
main recognition complex of 40 kDa (TRC40) that had been
identi1047297ed through biochemical strategies in mammalian
cell-free assays as a major interaction partner for newly syn-
thesized tail-anchored proteins (Stefanovic and Hegde
2007 Favaloro et al 2008) Subsequent synthetic genetic
array analyses and biochemical approaches in yeast (Jonikas
et al 2009 Battle et al 2010 Chang et al 2010 Chartron
et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models
for the GET targeting pathway in yeast suggest that a Sgt2ndash
Get4ndashGet5 subcomplex loads tail-anchored substrates onto
the targeting factor Get3 (Figure 1c) The Get3-bound
substrate then delivers these newly synthesized proteins
to an integral membrane Get1 Get2 complex In an ATP-
dependent process Get3 in association with Get1 Get2
then inserts the hydrophobic segment to span across the
ER membrane bilayer (Shao and Hegde 2011) Although
structural and biochemical studies are rapidly advancing
our understanding of the GET-dependent targeting path-
way the mechanisms by which tail-anchored proteins are
inserted into ER membrane bilayer remain to be de1047297ned
Maturation of secretory proteins in the ER signal sequence processing
For the many secretory proteins that contain an N-terminal
signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-
age site during translocation through the Sec61 complex
(Figure 2a) The SPC consists of four polypeptides termed
Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau
et al 1991) Spc3 and Sec11 are essential integral mem-
brane proteins that are required for signal sequence cleav-
age activity with the Sec11 subunit containing the protease
active site (Fang et al 1997 Meyer and Hartmann 1997)
Based on structural comparisons with E coli leader pepti-
dase the active site of SPC is thought to be located very near
the lumenal surface of the ER membrane and presumably
close to translocon exit sites The Spc1 and Spc2 subunits
are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-
cessed precursors of secretory proteins in vivo (Fang et al
1996) and are required for full enzymatic activity of the SPC
in vitro (Antonin et al 2000) Interestingly Spc2 is detected
in association with the Sbh1 subunit of the Sec61 complex
and is thought to physically link the SPC and Sec61 complex
(Antonin et al 2000) Given that SEC11 was identi1047297ed in
the original SEC mutant screen as required for ER-to-Golgi
transport of secretory proteins signal sequence cleavage is
regarded as an essential step for maturation of secretory
proteins that contain N-terminal signal sequences
Maturation of secretory proteins in the ER protein glycosylation
In addition to signal sequence cleavage attachment of
asparagine-linked oligosaccharide to nascent glycopro-
teins occurs concomitantly with polypeptide translocation
through the Sec61 pore (Figure 2b) The addition of core
oligosaccharides to consensus Asn-X-SerThr sites in transit-
ing polypeptides is catalyzed by the oligosaccharyltrans-
ferase (OST) enzyme OST is composed of eight integral
membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4
Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-
plex with the Sec61 translocon (Kelleher and Gilmore
2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before
addition of N-linked oligosaccharide demonstrating the se-
quential stages of polypeptide translocation signal sequence
cleavage and N-linked glycosylation (Chen et al 2001) The
Stt3 subunit is critical for catalytic activity and in addition to
Stt3 most of the OST subunits are required for cell viability
indicating a critical role for N-linked glycosylation in matu-
ration of secretory proteins OST transfers a 14-residue oli-
gosaccharide core en bloc to most (but not all) Asn-X-Ser
388 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 728
Thr sites in transiting polypeptides The 14-residue oligosac-
charide core is assembled on the lipid-linked carrier mole-
cule dolichylpyrophosphate in a complex multistep pathway
(Burda and Aebi 1999)
The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances
mutation of single and multiple sites within a given protein
produces only mild consequences Hydrophilic N-linked
glycans in1047298uence thermodynamic stability and solubility of
proteins and in the context of nascent secretory proteins
in the ER the N-linked structure is also thought to be an
integral part of a system that assists in protein folding and
quality control to manage misfolded glycoproteins (Schwarz
and Aebi 2011) This quality control process will be explored
further after covering other folding and post-translational
modi1047297cation events in secretory protein maturation
In addition to N-linked glycosylation some secretory
proteins undergo O-linked glycosylation through attach-
ment of mannose residues on SerThr amino acids by
protein O-mannosyltransferases (Pmts) Saccharomyces cer-
evisiae contains a family of seven integral membrane man-
nosyltranferases (Pmt1ndashPmt7) that covalently link mannose
residues to SerThr residues using dolichol phosphate man-
nose as the mannosyl donor (Orlean 1990 Willer et al
2003) Both O-linked mannose residues and N-linked core
oligosaccharides added in the ER are extended in the Golgi
complex by the nine-membered KRE2 MNT1 family of man-
nosyltranferases that use GDP-mannose in these polymeri-
zation reactions (Lussier et al 1997ab) O-linked mannosyl
modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall
integrity as well as normal morphogenesis (Strahl-Bolsinger
et al 1999) The role of O-linked glycosylation in ER quality
control processes remains unclear although investigators
have reported in1047298uences of speci1047297c pmt mutations on turn-
over rates of misfolded glycoproteins (Harty et al 2001
Vashist et al 2001 Hirayama et al 2008 Goder and Melero
2011) and the PMT genes are upregulated by activation of
the UPR (Travers et al 2000)
Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition
Approximately 15 of proteins that enter the secretory
pathway are post-translationally modi1047297ed on their C termi-
nus by addition of a lipid-anchored glycosylphosphatidyli-
nositol (GPI) moiety The synthesis and attachment of GPI
anchors occur in the ER through a multistep pathway that
depends on 20 gene products (Orlean and Menon 2007)
GPI synthesis and attachment are essential processes in
yeast and GPI anchored proteins on the cell surface are
thought to play critical roles in cell wall structure and cell
morphology (Leidich et al 1994 Pittet and Conzelmann
2007) As with assembly of the N-linked core oligosaccha-
ride the GPI anchor is fully synthesized as a lipid anchored
precursor and then transferred to target proteins en bloc by
the GPI transamidase complex (Fraering et al 2001) The
GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent
linkage to what becomes the terminal amino acid (termed the
v residue) and removal of the 30-amino-acid C-terminal
GPI signal sequence (Udenfriend and Kodukula 1995) Bio-
informatic approaches are now reasonably effective in pre-
dicting GPI anchored proteins These algorithms scan for
open reading frames that contain an N-terminal signal se-
quence and a C terminus that consists of an v residue
bracketed by 10 residues of moderate polarity plus a hy-
drophobic stretch near the C terminus of suf 1047297cient length
to span a membrane bilayer (Eisenhaber et al 2004) GPI
precursor proteins that do not receive GPI-anchor addition
and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering
and Schekman 1996) and are probably retained through an
ER quality control mechanism
Maturation of secretory proteins in the ER disul 1047297 debond formation
Most secretory proteins contain disul1047297de bonds that form
when nascent polypeptides are translocated into the oxidiz-
ing environment of the ER lumen A family of protein-
Figure 2 Folding and matura-
tion of secretory proteins A se-
ries of covalent modi1047297cations
and folding events accompany
secretory protein biogenesis in
the ER (A) Signal peptidase com-
plex consisting of Spc1Spc2
Spc3Sec11 cleaves hydrophobic
signal sequences during polypep-
tide translocation (B) Coincident
with polypeptide translocationand signal sequence cleavage
N-linked core-oligosaccharide is
attached to consensus N-X-ST
sites within the transiting poly-
peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly
catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose
residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1
For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation
Early Events in Protein Secretion 389
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 828
disul1047297de isomerases that contain thioredoxin-like domains
catalyze the formation reduction and isomerization of
disul1047297de bonds to facilitate correct protein folding in the
ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-
tein disul1047297de isomerase that is required for formation of
correct disul1047297de bonds in secretory and cell surface proteins
(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains
oxidizing equivalents for disul1047297de formation from the es-
sential 1047298
avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and
Pdi1 form the major pathway for protein disul1047297de bond
formation by shuttling electrons between Ero1 Pdi1 and
substrate proteins (Tu and Weissman 2002 Gross et al
2006) In reconstituted cell-free reactions FAD-linked Ero1
can use molecular oxygen as the electron acceptor to drive
Pdi1 and substrate protein oxidation The electron acceptor(s)
used by Ero1 in vivo remain to be fully characterized (Hatahet
and Ruddock 2009)
In addition to Pdi1 yeast express four other nonessential
ER-localized protein disul1047297de isomerase homologs Mpd1
Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant
forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In
addition to oxidoreductase activity Pdi1 can act as a molec-
ular chaperone in protein folding even for proteins that lack
disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)
More recently Pdi1 and other members of this family were
reported to interact with components of the ER folding ma-
chinery including calnexin (Cne1) and Kar2 (Kimura et al
2005) as well as the quality control mannosidase enzyme
Htm1 (Gauss et al 2011) Growing evidence indicates that
this family of protein disul1047297de isomerases contains different
domain architectures (Vitu et al 2008) to dictate interac-
tions with speci1047297c ER-chaperone proteins and thus shepherd
a broad range of client proteins into folded forms or into ER-
associated degradation pathways (Figure 2d)
Glucosidase mannosidase trimming and protein folding
The initial 14-residue N-linked core oligosaccharide that is
attached en bloc to nascent polypeptides is subsequently
processed by glycosylhydrolases in a sequential and protein
conformation-dependent manner to assist protein folding
and quality control in the ER lumen (Helenius and Aebi
2004) The Glc3Man9GlcNAc2 glycan which comprises the
N-linked core is rapidly processed by glucosidase I (Gls1
Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove
the three terminal glucose residues and generate Man9-
GlcNAc2 Molecular chaperones collaborate in protein fold-
ing during these glucose-trimming events and Rot1 alone
has been shown to possess a general chaperone activity
(Takeuchi et al 2008) In many cell types a calnexin-
dependent folding cycle operates to iteratively fold and
monitor polypeptide status through the coordinated activi-
ties of glucosidase I glucosidase II UDP-glucoseglycopro-
tein glucosyltransferase (UGGT) and calnexin (Cne1) After
removal of terminal glucose residues by the glucosidase
enzymes UGGT can add back a terminal glucose to the
glycan if the polypeptide is not fully folded to generate the
Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of
an unfolded protein binds to calnexin which keeps the na-
scent polypeptide in an iterative folding cycle Once fully
folded UGGT does not act after glucosidase II and the na-
scent protein exits the cycle (Helenius and Aebi 2004) This
calnexin cycle operates in many eukaryotes but it is cur-
rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like
protein) do not produce strong delays in biogenesis of se-
cretory proteins but are known to produce defects in bio-
synthesis of cell wall b-16-glucan (Shahinian and Bussey
2000) Although a precise molecular understanding of the
calnexin cycle components in yeast folding remains to be
determined there are clear genetic (Takeuchi et al 2006
Costanzo et al 2010) and biochemical (Xu et al 2004
Kimura et al 2005) interactions that indicate a coordinated
role for these factors in protein folding
In addition to the glucose trimming of core oligosaccha-
ride two additional ER-localized mannosidase enzymes
termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)
Mns1 and Htm1 are related enzymes with distinct speci1047297c-
ities Mns1 removes the terminal mannosyl residue of the B
branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported
from the ER (Jakob et al 1998) Htm1 is thought to act after
Mns1 on terminally misfolded proteins (or misfolded pro-
teins that have lingered in the ER folding cycle for too long)
to remove the outermost mannosyl residue from the C
branch of the glycan to generate Man7GlcNAc2 (Clerc
et al 2009) This form of the glycan is then recognized by
the ER lectin Yos9 and targets misfolded proteins for ER-
associated degradation (Carvalho et al 2006 Denic et al
2006) Although Mns1- and Htm1-de1047297cient cells appear to
transport folded secretory proteins at normal rates both
display signi1047297cant delays in turnover of terminally misfolded
glycoproteins (Jakob et al 1998 2001) which serves to
highlight an important role for mannosidase activity in ER
quality control
Folding of nascent polypeptides throughout transloca-
tion and within the ER is also managed by Hsp70 ATPase
systems which handle partially folded intermediates In
general Hsp70 proteins hydrolyze ATP when binding to
exposed hydrophobic stretches in unfolded polypeptides
to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70
ATPase cycle governed by speci1047297c DnaJ-like proteins that
stimulate ATP hydrolysis and nucleotide exchange factors that
drive ADP release (Hartl 1996 Bukau and Horwich 1998) In
yeast the Hsp70 Kar2 plays a prominent role in ER folding in
concert with the related Hsp70 protein Lhs1 (Rose et al
1989 Baxter et al 1996 Brodsky et al 1999 Steel et al
2004) For Kar2 the known DnaJ-like stimulating factors
include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995
390 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 928
Nishikawa and Endo 1997) whereas the GrpE family mem-
ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as
nucleotide exchange factors (Hale et al 2010) Complexity in
regulating the Kar2 ATPase cycle probably re1047298ects the range of
unfolded substrates that Kar2 must handle in maintaining ER
homeostasis and there are likely to be additional factors that
couple Kar2 activity to other speci1047297c ER processes As mentioned
above Kar2 chaperone activity is tightly linked with the PDI
calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation
(ERAD) pathways to dispose of terminally misfolded proteins
(Nishikawa et al 2001) Although our understanding of Kar2
biochemical activity is advanced the coordinated control of
Kar2-dependent folding and modi1047297cation cycles in the context
of an ER lumenal environment remains a challenging area
ERAD of misfolded and unassembled proteins proceeds
through a series of pathways that remove targeted proteins
from the ER for ubiquitin- and proteasome-dependent deg-
radation in the cytoplasm ERAD is thought to play a key
role in ER homeostasis and cellular physiology Since these
pathways divert misfolded secretory proteins from their
routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to
excellent recent reviews (Vembar and Brodsky 2008 Smith
et al 2011)
Control of ER homeostasis by the Unfolded Protein Response
Much of the folding and biogenesis machinery in the ER is
under a global transcriptional control program referred to
as the UPR The yeast UPR is activated by an increase in
the level of unfolded proteins in the ER which can be
experimentally induced by treatment with inhibitors of
ER protein folding (eg tunicamycin dithiothreitol) or by
overexpression of terminally misfolded proteins (Bernales
et al 2006) Regulation of the UPR was initially examined
through identi1047297cation of a 22-nucleotide segment in the
KAR2 promoter region termed the unfolded protein re-
sponse element (UPRE) which was required for UPR ac-
tivation of Kar2 expression Fusion of this KAR2 promoter
element to a lacZ reporter provided an elegant screen for
gene mutations that blunted UPR reporter expression (Cox
et al 1993 Mori et al 1993) Genetic screening led to the
discovery that IRE1 HAC1 and RLG1 were required for
a robust UPR under ER stress conditions (Cox and Walter
1996 Sidrauski et al 1996) Further studies revealed that
IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain
that together are thought to serve as readout on unfolded
protein levels HAC1 encodes a basic leucine zipper tran-
scription factor that binds to UPRE-containing segments of
DNA and induces their expression (Cox and Walter 1996)
Surprisingly RLG1 encodes a tRNA ligase that is required for
the nonconventional splicing of HAC1 pre-mRNA Structural
and mechanistic dissection of these core components is now
advanced Current models indicate that the Ire1 lumenal
domain interacts with Kar2 and unfolded proteins to sense
protein folding status (Bertolotti et al 2000 Pincus et al
2010 Gardner and Walter 2011) When unfolded proteins
accumulate in the ER Ire1 forms oligomers that activate the
cytoplasmic kinase and ribonuclease domains Activated
Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove
a nonconventional intron and this splicing intermediate is
then ligated by the Rlg1 ligase to produce mature HAC1
mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target
genes (Bernales et al 2006)
In addition to Kar2 the UPR was known to induce other
ER folding components including Pdi1 and Eug1 (Cox et al
1993 Mori et al 1993) To comprehensively assess the tran-
scriptional pro1047297le of the yeast UPR DNA microarray analysis
was powerfully applied to monitor mRNA levels under ER
stress conditions (Travers et al 2000) Comparing transcrip-
tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR
induction revealed 381 genes that passed stringent criteria
as UPR targets Not surprisingly 10 genes involved in ER
protein folding were identi1047297ed as UPR targets and included
JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-
tion and ER-associated degradation were induced Perhaps
more surprisingly 19 genes involved in lipid and inositol
metabolism as well as 16 genes encoding proteins that func-
tion in vesicle traf 1047297cking between the ER and Golgi were
upregulated by the UPR These 1047297ndings highlight a global
role for the UPR in regulating ER homeostasis through bal-
ancing ER lipid and protein biosynthetic rates In the context
of cellular physiology the UPR is now thought to serve a cen-
tral role in sensing and integrating secretory pathway func-
tion to 1047297nely tune ER capacity in response to cellular
demands (Walter and Ron 2011)
Transport From the ER Sculpting and Populatinga COPII Vesicle
Once secretory proteins have completed their synthesis and
modi1047297cation regimes they become competent for forward
traf 1047297c through the secretory pathway a process mediated
by a series of transport vesicles that bud off from one
compartment traverse the cytoplasm and fuse with a down-
stream organelle (Figure 3) ER-derived vesicles are created
by the COPII coat that like other coat protein complexes is
charged with the dual tasks of creating a spherical transport
vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical
characterization of this process 1047297rst from complex mi-
crosomal membranes using puri1047297ed COPII coat proteins
(Barlowe et al 1994) then in more reduced form from syn-
thetic liposomes (Matsuoka et al 1998b) and subsequently
at the structural level through cryo-EM (Stagg et al 2006)
and X-ray crystallography (Bi et al 2002 Fath et al 2007)
has been remarkably fruitful in de1047297ning the molecular basis
of these events What has emerged is an elegant mechanism
Early Events in Protein Secretion 391
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1028
whereby the minimal COPII machinery composed of 1047297 ve
proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces
to ful1047297ll these multiple functions However recent insights
into how this process is regulated suggest there is still much
to learn about coat dynamics in the cell and the precise
physical basis for various steps including membrane scission
during vesicle release vesicle uncoating and the formation
of large transport carriers capable of shuttling large cargoes
Structure and assembly of the COPII coat
COPII coat assembly (Figure 3) is initiated by the local re-
cruitment and activation of the small G protein Sar1
(Nakano and Muramatsu 1989 Barlowe et al 1993) upon
exchange of GDP for GTP catalyzed by an ER membrane
protein the guanine nucleotide exchange factor (GEF)
Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-
ing on Sar1 exposes an amphipathic a-helix that likely
induces initial membrane curvature by locally expanding
the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee
et al 2005) GTP-bound membrane-associated Sar1 sub-
sequently recruits the heterodimeric complex of Sec23
and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-
activating protein (GAP) for Sar1 (Yoshihisa et al 1993)
contributing a catalytic arginine residue analogous to GAP
stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-
taining multiple independent domains that interact directly
with speci1047297c sorting signals on various cargo proteins (Miller
et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23
Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-
tetrameric complex of Sec13 and Sec31 (Matsuoka et al
1998b) Sec31 also contributes to the GTPase activity of
the coat by stimulating the GAP activity of Sec23 (Antonny
et al 2001 Bi et al 2007) Thus the fully assembled coat is
composed of two distinct layers the ldquoinnerrdquo membrane
proximal layer of Sar1 Sec23 Sec24 that intimately asso-
ciates with lipid headgroups (Matsuoka et al 2001) and
contributes cargo-binding function and the ldquoouterrdquo mem-
brane distal layer composed of Sec13 Sec31 Both layers
contribute to the catalytic cycle of Sar1 and endowing
maximal GTPase activity when the coat is fully assembled
(Antonny et al 2001)
Our mechanistic understanding of COPII coat action has
been signi1047297cantly enhanced by the structural characteriza-
tion of the different coat components A structure of the
Sec23 Sec24 dimer showed a bow-tie shaped assembly with
a concave face that is presumed to lie proximal to the mem-
brane and is enriched in basic amino acids (Bi et al 2002)
These charged residues may facilitate association with the
acidic phospholipid headgroups of the ER membrane Sub-
sequent structural genetic and biochemical analyses of
Sec24 revealed multiple discrete sites of cargo interaction
dispersed around the perimeter of the protein (Miller et al
2003 Mossessova et al 2003) Structural analysis of the
outer coat was facilitated by the observation that under
some conditions the puri1047297ed coat proteins can self-assemble
into ldquocagesrdquo of the approximate size of a COPII vesicle
(Antonny et al 2003) Further experiments using mamma-
lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII
cage which forms a lattice-like structure with geometry dis-
tinct from that of the clathrin coat (Stagg et al 2006) Het-
erotetrameric Sec13 Sec31 complexes form straight rods
known as ldquoedgerdquo elements four of which come together at
ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-
quent crystal structures of Sec13 and a portion of Sec31
revealed an unexpected domain arrangement within the
edge element whereby Sec31 forms both the dimerization
Figure 3 Coat assembly drives
vesicle formation Both the COPII
(left) and COPI (right) coats are
directed in their assembly by
small GTPases of the ArfSar1
family In the COPII coat Sar1
is activated by its guanine nu-
cleotide exchange factor (GEF)
Sec12 which localizes to the ER
membrane Activated Sar1ndashGTP
recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-
ner coatrdquo layer and provides the
cargo-binding function A heter-
otetramer of Sec13Sec31 is sub-
sequently recruited forming the
ldquoouter coatrdquo and polymerizing
into a lattice-like structure that
drives membrane curvature In
the COPII cage formed by Sec13
Sec31 four molecules of Sec31
assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is
driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology
to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions
of three b-propeller domains of Sec27 (inset)
392 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1128
interface along the edge element and the vertex assembly
unit with Sec13 sandwiched between these structural ele-
ments (Fath et al 2007) However the fragment of Sec31
that 1047297ts well into the density of the cryo-EM structure
represents only about half of the protein an additional
proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound
to Sar1 Sec23 has yielded great insight into the mecha-
nism of GAP activity whereby the active fragment of Sec31
lies along the membrane-distal surface of Sec23 Sar1 and
optimizes the orientation of the catalytic histidine of Sar1
(Bi et al 2007)
The ability of Sec13 Sec31 to assemble into a spherical
structure that matches closely the size of a COPII vesicle
suggests that the primary membrane bending force may
come from the scaffolding effect of this structure on the
ER membrane Indeed when the curvature-inducing amphi-
pathic helix of Sar1 is replaced with an N-terminal histidine
tag to drive recruitment to Ni-containing liposomes subse-
quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-
1047297cient to drive the generation of spherical buds that remain
attached to the donor liposome (Lee et al 2005) Thus an
additional function of the Sar1 helix is to drive vesicle scis-
sion a model supported by experiments that link GTPase
activity to vesicle release in a manner analogous to that
proposed for dynamin (Pucadyil and Schmid 2009 Kung
et al 2012) Although the concave face of Sec23 Sec24
may also contribute to membrane curvature it has been
suggested that the relatively paltry dimer interface between
these two molecules is not robust enough to impart curva-
ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and
Sec23 Sec24 may participate in membrane curvature the
majority of membrane bending force likely comes from
Sec13 Sec31 Indeed recent genetic and biochemical
experiments support this model Sec31 likely forms all the
contacts needed to make the COPII cage (Fath et al 2007)
with Sec13 providing structural rigidity to the cage edge
element to overcome the membrane bending energy of
a cargo-rich membrane (Copic et al 2012)
Cargo capture stochastic sampling vs direct and indirect selection
The fundamental function of vesicles is to ensure directional
traf 1047297c of protein cargoes making cargo capture an in-
tegral part of coat action To some extent cargo can enter
into vesicles in a nonspeci1047297c manner known as bulk 1047298ow
whereby stochastic sampling of the ER membrane and
lumen occurs during vesicle formation capturing local
molecules by chance Although this mode of transport could
traf 1047297c some abundant cargoes the random nature of this
process cannot explain the ef 1047297ciency with which some ER
export occurs In particular some cargoes are dramatically
enriched in vesicles above their prevailing concentration in
the ER suggesting a more ef 1047297cient and selective packaging
process Although the concentrative mode of cargo selection
has gained favor in the last decade recent experiments
reevaluating the potential for bulk 1047298ow to explain forward
traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-
cially with respect to abundant nonessential proteins where
the ef 1047297ciency of secretion may not be central to cellular
viability (Thor et al 2009)
Selective enrichment of cargo in transport vesicles via
speci1047297c sorting signals is a common paradigm in intracellu-
lar protein traf 1047297cking 1047297rst characterized in endocytosis
Deciphering a similar mode of transport for the entire
spectrum of cargoes handled by the COPII coat however
has been hindered by the absence of a single common signal
used by the entire secretome Instead multiple signals seem
to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse
cargoes Such signals range from simple acidic peptides
(Malkus et al 2002) to folded epitopes (Mancias and Goldberg
2007) and can act either by interacting directly with the
COPII coat or by binding to a cargo adaptor that links them
to the coat indirectly (Figure 4) (Dancourt and Barlowe
2010)
Genetic biochemical and structural data support Sec24
as the cargo binding adaptor for the COPII coat forming
Figure 4 Cargo selection can be direct or indirect Selec-
tive cargo capture during vesicle formation can occur via
direct interaction of cargo molecules with the COPI and
COPII coats ER export signals (eg DxE LxxLE and
YxxNPF) interact directly with Sec24 to facilitate capture
into COPII vesicles Similarly dilysine and diaromatic sig-
nals mediate interaction with the COPI coat to direct ret-
rograde traf1047297c back to the ER Soluble secretory proteins
may be captured indirectly via speci1047297c cargo receptors that
serve to recognize the transport-competent cargo and link
it to the coat Erv29 is the cargo receptor for many soluble
secretory proteins Soluble ER residents are returned back
to the ER via a similar cargo receptor system driven by
Erd2 which recognizes HDEL signals Membrane proteins
may also require cargo adaptor proteins such as Erv14 and
Rer1 although the basis for cargo recognition is not as
well de1047297ned
Early Events in Protein Secretion 393
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1228
a relatively static platform that has multiple binding sites for
interaction with distinct sorting signals The so-called A site
binds the SNARE Sed5 via a NPF motif (Mossessova et al
2003 Miller et al 2005) the B site is most diverse recog-
nizing acidic sorting signals such as those found on the
SNARE Bet1 the Golgi membrane protein Sys1 and un-
known signals on additional cargoes (Miller et al 2003
Mossessova et al 2003) the C site binds a folded epitope
formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of
binding sites is further expanded by the presence of addi-
tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-
teins (Roberg et al 1999 Kurihara et al 2000 Peng et al
2000) Sec24ndashcargo interactions are in general fairly low
af 1047297nity (Mossessova et al 2003) which is compatible with
the transient nature of the association of cargo with coat
proteins must bind during vesicle formation but must also be
released prior to vesicle fusion to allow coat recycling and
exposure of fusogenic domains The possibility remains that
additional layers of regulation impact coat dissociation from
cargo molecules after vesicle release Sec23 is both ubiquiti-
nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-
pling of coat from cargo
Some cargoes by topology or preference do not interact
directly with Sec24 but instead use adaptorreceptor pro-
teins to link them to the coat indirectly (Dancourt and
Barlowe 2010) Some of these adaptors likely function as
canonical receptors binding to their ligands in one compart-
ment and simultaneously interacting with Sec24 to couple
cargo with coat then releasing their ligand in another com-
partment perhaps as the result of a change in ionic strength
or pH of the acceptor organelle (Figure 3) Although their
precise mechanisms of ligand binding and release remain to
be fully explored such receptors include Erv29 which medi-
ates traf 1047297c of soluble secretory proteins like pro-a-factor and
CPY (Belden and Barlowe 2001) and Emp46 Emp47 which
are homologous to the mammalian ERGIC-53 family of pro-
teins that mediate traf 1047297c of coagulation factors (Sato and
Nakano 2002) Other receptors function to enrich vesicles
with membrane protein cargoes The p24 proteins Emp24
Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-
port of GPI-anchored proteins whose lumenal orientation
precludes direct coupling to the COPII coat (Belden and
Barlowe 1996 Muniz et al 2000 Belden 2001) Others like
Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14
(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-
brane proteins that have cytoplasmically oriented regions
but either do not contain ER export signals or require addi-
tional af 1047297nity or organization to achieve ef 1047297cient capture
The requirement for receptors for such transmembrane car-
goes remains unexplained but may derive from the ancestral
history of the cargoes whereby previously soluble proteins
became membrane anchored as a result of gene fusion events
(Dancourt and Barlowe 2010) Alternatively the receptor
proteins may provide additional functionality required for
ef 1047297cient ER egress like a chaperoning function that would
protect the long transmembrane domains of plasma mem-
brane proteins from the relatively thinner lipid bilayer char-
acteristic of the ER (Sharpe et al 2010) Indeed some cargo
proteins have speci1047297c chaperoning needs with ER resi-
dent proteins that are not themselves captured into COPII
vesicles likely functioning to promote assembly and folding
of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-
rect folding and quaternary assembly which is itself a pre-
requisite for COPII capture (Ljungdahl et al 1992 Kuehn
et al 1996 Gilstring et al 1999 Kota et al 2007)
Regulation of COPII function GTPase modulationcoat modi 1047297 cation
The GTPase activity of the coat is the primary mode of
regulation known to govern initiation of coat assembly
disassembly through canonical GEF and GAP activities of
Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al
1993) respectively but also contributing to additional func-
tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005
Lee et al 2005) Unlike other coat systems the COPII coat
uses a combinatorial GAP activity that is provided by com-
ponents of the coat themselves Sec23 (Yoshihisa et al
1993) and Sec31 (Antonny et al 2001) The effect of this
autonomous GAP in minimal systems is that as soon as the
coat fully assembles GTP is hydrolyzed and the coat is rap-
idly released (Antonny et al 2001) creating a paradox as to
how coat assembly might be sustained for a suf 1047297cient length
of time to generate vesicles One solution to this conundrum
is that constant Sec12 GEF activity feeds new coat elements
into a nascent bud (Futai et al 2004 Sato and Nakano
2005) coat release from the membrane might also be
delayed by the increased af 1047297nity afforded by cargo proteins
(Sato and Nakano 2005) However recent 1047297ndings suggest
that a GAP inhibitory function contributed by the peripheral
ER protein Sec16 also modulates the activity of the coat
(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is
a large essential protein that associates with the cytoplas-
mic face of the ER membrane at ERES (Espenshade et al
1995 Connerly et al 2005) It interacts with all of the COPII
coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and
is thus thought to scaffold andor organize coat assembly at
these discrete domains (Supek et al 2002 Shindiapina and
Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect
of Sec31 probably by preventing Sec31 recruitment to
Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory
effect of Sec16 was diminished in the context of a point muta-
tion in Sec24 (Kung et al 2012) raising the tantalizing possi-
bility that cargo engagement by Sec24 could trigger interaction
with Sec16 to inhibit the full GTPase activity of the coat in such
a manner that a vesicle is initiated around a cargo-bound com-
plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)
394 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1328
Another poorly explored aspect of COPII regulation is
post-translational modi1047297cation of the coat Sec23 is a target
for ubiquitination and is seemingly rescued from degrada-
tion by the action of the ubiqutin protease complex Bre5
Ubp3 (Cohen et al 2003) Whether this activity only con-
trols expression levels of the protein or contributes more
subtly to regulate proteinndashprotein interactions remains to
be tested Furthermore the potential ubiquitination of other
COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target
for a speci1047297c monoubiquitination event that is important for
ER export of collagen 1047297bers (Jin et al 2012) Whether yeast
Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin
ligases and how such a modi1047297cation might in1047298uence coat
action perhaps by contributing to the structural integrity
of the coat to drive membrane bending around rigid car-
goes remains to be tested Like ubiquitination the role of
coat phosphorylation is only starting to be explored It has
long been known that Sec31 is a phosphoprotein and that
dephosphorylation speci1047297cally impacted vesicle release
(Salama et al 1997) However despite the many sites of
Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-
cations remains unclear In contrast progress has recently
been made in understanding phosphorylation of Sec23
and how this event probably in1047298uences the directionality
of vesicle traf 1047297c by controlling sequential interactions with
different Sec23 partners (Lord et al 2011) It is tempting to
speculate that similar phosphorylation of Sec24 might also
regulate coat displacement from cargo molecules to further
promote coat release and expose the fusogenic SNARE pro-
teins that would otherwise be occluded by their interaction
with the coat Indeed at least partial uncoating of COPII
vesicles is required for fusion to ensue since when GTP hy-
drolysis is prevented vesicles fail to fuse (Barlowe et al
1994) Whether additional proteinndashprotein interactions or
post-translational modi1047297cations contribute to coat shedding
remains to be seen
Higher-order organization of vesicle formation
Although the minimal COPII coat can drive vesicle forma-
tion from naked liposomes (Matsuoka et al 1998b) this
process in vivo is likely tightly regulated to enable both ef-
1047297cient vesicle production and adaptability to suit the secre-
tory burden of the cell (Farhan et al 2008) In part this
regulation occurs at the level of the subdivision of the ER
into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-
selves and accessory proteins such as Sec16 and in some
cells Sec12 (Rossanese et al 1999 Connerly et al 2005
Watson et al 2006) ERES are located throughout the ER
with a seemingly random distribution that may in fact cor-
respond to regions of high local curvature induced by the ER
membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al
2012) In related yeasts these sites are dynamic with the
ability to form de novo fuse and divide (Bevis et al 2002)
Although the precise mechanisms that regulate the steady
state distribution and size of these domains remain unclear
activity of both Sec12 and Sec16 seems to play a role
(Connerly et al 2005) as does the lipid composition of
the ER (Shindiapina and Barlowe 2010) In mammalian
cells misfolded proteins that are incompetent for forward
traf 1047297c are excluded from ERES (Mezzacasa and Helenius
2002) and this also seems to be true for some proteins
in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which
are not concentrated at ERES but instead remain dispersed
within the bulk ER (Castillon et al 2009)
Vesicle Delivery to the Golgi
After release of COPII vesicles from ER membranes tethering
and fusion machineries guide ER-derived vesicles to Golgi
acceptor membranes through the action of over a dozen
gene products (Figure 5) Although ER ndashGolgi transport
can be separated into biochemically distinct stages using
cell-free assays evidence suggests that these events may
be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered
to Golgi membranes through the action of the Ypt1 GTPase
and tethering proteins Uso1 and the transport protein par-
ticle I (TRAPPI) complex Membrane fusion between vesicle
and Golgi acceptor membranes is then catalyzed through
assembly of SNARE protein complexes from the apposed
membrane compartments How the budding tethering
and fusion events are coordinated in cells remains an open
question although genetic biochemical and structural
studies have advanced our understanding of underlying
molecular mechanisms in vesicle tethering and membrane
fusion described below
Vesicle tethering
Initial cell free transport assays coupled with genetic ap-
proaches placed ER ndashGolgi transport requirements into
distinct vesicle budding and vesicle consumptionfusion
stages (Kaiser and Schekman 1990 Rexach and Schekman
1991) Ypt1 identi1047297ed as a founding member of the Rab
family of GTPases was implicated in the vesicle targeting
stage in the ER ndashGolgi transport pathway (Schmitt et al
1988 Segev et al 1988 Baker et al 1990) In reconstituted
vesicle fusion reactions Ypt1 was found to act in concert
with the extended coil-coiled domain protein Uso1 to tether
COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII
vesicles could be tethered to and sedimented with washed
Golgi acceptor membranes upon addition of puri1047297ed Uso1
Interestingly the Uso1- and Ypt1-dependent tethering stage
does not appear to require the downstream SNARE protein
fusion machinery (Sapperstein et al 1996 Cao et al 1998)
In addition to the extended structure of Uso1 which is
predicted to span a distance of 180 nm (Yamakawa et al
1996) the multisubunit TRAPPI complex is required for
Early Events in Protein Secretion 395
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1428
COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays
revealed that TRAPPI can also function to physically link
COPII vesicles to Golgi membranes (Sacher et al 2001)
Structural analyses show that TRAPPI is a 170-kDa particle
consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31
and Trs33) that assemble into a 1047298at bilobed arrangement
with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al
2006) Bet3 can bind directly to Sec23 and with TRAPPI
peripherally bound to membranes this activity is thought
to link partially coated COPII vesicles to Golgi acceptor
membranes (Cai et al 2007) In a recent study the Golgi-
associated Hrr25 kinase was reported to phosphorylate
Sec23 Sec24 and regulate interactions between Sec23 and
TRAPPI to control directionality of anterograde transport (Lord
et al 2011) Moreover TRAPPI functions as a GEF for Ypt1
in a manner that is thought to generate activated Ypt1 on
the surface of Golgi acceptor membranes andor COPII
vesicles (Jones et al 2000 Wang et al 2000 Lord et al
2011) A subassembly of TRAPPI consisting of Bet3 Bet5
Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-
change by stabilizing an open form of this GTPase (Cai et al
2008) TRAPPI does not appear to interact directly with
Uso1 although Ypt1 activation could serve to coordinate
the long-distance tethering mediated by Uso1 with a closer
TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known
but current models suggest that this multisubunit complex
links COPII vesicles to the cis-Golgi surface and serves as a
central hub in coordinating vesicle tethering with SNARE-
mediated membrane fusion
Genetic and biochemical evidence indicate that other
coiled-coil domain proteins also act in COPII vesicle tether-
ing andor organization of the early Golgi compartment in
yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi
membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed
Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential
but deletion of either protein reduces COPII vesicle tether-
ing and transport levels in cell-free assays and the grh1 D
and bug1 D mutants display negative genetic interactions
with thermosensitive ypt1 and uso1 mutants (Behnia et al
2007) These 1047297ndings suggest a redundant network of
coiled-coil proteins that act in tethering vesicles and orga-
nizing the cis-Golgi compartment Indeed additional coiled-
coil proteins including Rud3 and Coy1 localize to cis-Golgi
membranes and are implicated in organization of the cis-
Golgi and interface with COPII vesicles (VanRheenen et al
1999 Gillingham et al 2002 2004) Although some double
deletion analyses have been performed with these genes
multiple deletions may be required to severely impact this
redundant network
SNARE protein-dependent membrane fusion
Fusion of tethered COPII vesicles with cis-Golgi membranes
depends on a set of membrane-bound SNARE proteins Sev-
eral lines of evidence indicate that the SNARE proteins
Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion
event in yeast (Newman et al 1990 Hardwick and Pelham
1992 Sogaard et al 1994 Cao and Barlowe 2000) The
SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-
tif which is typically adjacent to a C-terminal tail-anchored
membrane segment (Rothman 1994 Fasshauer et al 1998)
Cognate sets of SNARE proteins form stable complexes
through assembly of their SNARE motifs into parallel four-
helix coiled-coil structures (Hanson et al 1997 Sutton et al
1998) The close apposition of membranes that follows as-
sembly of SNARE complexes in trans is thought to drive
membrane bilayer fusion (Weber et al 1998) Structural
Figure 5 Vesicle tethering and fu-
sion Anterograde delivery of COPII-
coated vesicles is mediated by a
variety of tethering and fusion com-
plexes The TRAPP complex binds to
Sec23 on the surface of a COPII ves-
icle and mediates local activation of
the Rab family member Ypt1 Yptndash
GTP recruits downstream effectors
such as the long coiled-coil tether
Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-
ces TRAPP perhaps contributing to
coat shedding Removal of the coat
exposes the fusogenic SNARE pro-
teins which assemble to drive
membrane mixing In the retrograde
pathway COPI-coated vesicles em-
ploy the DSL1 complex composed
of Dsl1Sec39Tip20 to recognize
the incoming vesicle and coordinate
coat release and SNARE pairing
396 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1528
studies of the four-helix bundle reveal that the central or
ldquozero layerrdquo consists of ionic residues such that three of the
SNARE proteins contribute a glutamine residue and are
thus termed Q-SNARES whereas the fourth helix contains
an arginine residue and is known as the R-SNARE (Fasshauer
et al 1998 Sutton et al 1998) Further re1047297nement of the
Q-SNARE proteins based on sequence conservation iden-
ti1047297es each as a member of the Qa Qb or Qc subfamily
(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in
which three Q-SNARES (Qa Qb and Qc) and one R-SNARE
zipper together from the N-terminal side of the SNARE motif
toward the membrane (Sudhof and Rothman 2009) In
the case of COPII vesicle fusion with Golgi membranes
Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1
the Qc-SNARE and Sec22 the R-SNARE Furthermore this
SNARE set is suf 1047297cient to catalyze membrane fusion when
reconstituted into synthetic proteoliposomes (Parlati et al
2000)
In addition to Sed5 Bos1 Bet1 and Sec22 other regu-
latory factors are required to control fusion speci1047297city and
govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins
regulate distinct SNARE-dependent fusion events (Sudhof
and Rothman 2009) The SM family member Sly1 is re-
quired for fusion of COPII vesicles with Golgi membrane
in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-
tially identi1047297ed as a suppressor of loss of YPT1 function
when the gain-of-function SLY1-20 allele was isolated in
a selection for mutations that permit growth in the absence
of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5
and increases the 1047297delity of SNARE complex assembly be-
tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate
SNARE complexes (Peng and Gallwitz 2002) Crystallo-
graphic studies of Sly1 reveal a three-domain arch-shaped
architecture that binds a 45-amino-acid N-terminal domain
of Sed5 as observed for other SM protein interactions with
Qa-SNAREs (Bracher and Weissenhorn 2002) Working
models for Sly1 and SM protein function in general are
based on multiple binding modes wherein Sly1 initially
bound to the N terminus of Sed5 would subsequently bind
to other cognate SNARE proteins to regulate assembly and
ultimately to act as a clamp in stabilizing a trans-SNARE
complex (Furgason et al 2009 Sudhof and Rothman 2009)
After SNARE-mediated membrane fusion is complete
stable four-helix bundles of cis-SNARE complexes are now
present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-
ditional rounds of membrane fusion the general fusion fac-
tors Sec17 and Sec18 catalyze SNARE complex disassembly
(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18
belongs to the AAA family of ATPase chaperones and uses
the energy of ATP hydrolysis to separate stable cis-SNARE
complexes Sec17 is thought to recruit Sec18 to SNARE pro-
tein complexes and couples ATPase dependent disassembly
of cis-SNARE complexes (Bonifacino and Glick 2004) How
Sec17 Sec18-mediated disassembly is coordinated with
coat-dependent capture of SNARE proteins into vesicles
and Sly1-dependent assembly of trans-SNARE complexes
during fusion remain open questions
A concerted model for COPII vesicle tethering and fusion
Although distinct stages in vesicle tethering and fusion can
be de1047297ned through biochemical and genetic analyses these
are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-
subunit TRAPPI may serve as an organizational hub on cis-
Golgi membranes or vesicles to coordinate vesicle tethering
and fusion events TRAPPI interactions with the COPII
subunit Sec23 with the Ypt1 GTPase and potentially with
SNARE proteins (Jang et al 2002 Kim et al 2006) could
link tethering and fusion stages TRAPPI-activated Ypt1
could recruit Uso1 to Golgi membranes and as COPII
vesicles emerge from the ER Uso1 could forge a long-
distance link between newly formed vesicles and acceptor
membranes With tethered vesicles aligned to fusion sites
TRAPPI interactions with vesicle-associated Sec23 and Golgi
SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles
could transmit signals to the SNARE machinery by direct
contact or perhaps through generation of elevated levels of
activated Ypt1 The result of such a signal may be to disas-
semble cis-SNARE complexes or to generate a Sly1ndashSed5
conformation that promotes assembly of fusogeneic SNARE
complexes Assembly of trans-SNARE complexes would then
presumably lead to rapid hemifusion followed by bilayer
fusion and compartment mixing
Traf1047297c Within the Golgi
Transport through the Golgi complex
Newly synthesized secretory proteins arrive at the cis-Golgi
in COPII vesicles and after membrane fusion progress
through the Golgi complex Secretory cargo may receive
outer-chain carbohydrate modi1047297cations and proteolytic pro-
cessing in a sequential manner as cargo advances through
distinct Golgi compartments For glycoproteins the N-linked
core carbohydrate is extended by addition of a-16-mannose
residues in the cis-Golgi and by addition of a-12- and
a-13-mannose residues in the medial compartment Kex2-
dependent proteolytic processing of certain secretory cargo
occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-
activation of the thermosensitive sec18-1 allele (Graham and
Emr 1991 Brigance et al 2000) In support of this sequen-
tial organization distinct Golgi compartments can be visu-
alized through 1047298uorescence microscopy or immuno-EM
by monitoring components of the glycosylation and pro-
cessing machinery (Franzusoff et al 1991 Preuss et al 1992
Wooding and Pelham 1998 Rossanese et al 1999) However
genetic and morphological approaches have not uncovered
Early Events in Protein Secretion 397
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1628
a vesicle-mediated anterograde transport pathway through
distinct compartments of the yeast Golgi complex Instead
a model of cisternal maturation in which Golgi cisternae are
the anterograde carriers of secretory cargo is most consis-
tent with a range of experimental observations (Bonifacino
and Glick 2004) In the cisternal maturation model Golgi
cisterna containing nascent secretory cargo are formed at
the cis-face of the Golgi and mature into a medial and then
trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde
vesicles to preceding cisternae Indeed the dispersed orga-
nization of Golgi compartments in S cerevisiae are resolv-
able by 1047298uorescence microscopy and provided a powerful
test of the maturation model through live cell imaging of
cis- and trans-Golgi proteins labeled with different 1047298uores-
cent tags In such a dual labeled strain a cis-compartment
should be observed to change color to a trans-compartment
over the time period required for secretory cargo to transit
the Golgi complex Strikingly two independent research
groups using time resolved high resolution microscopy docu-
mented individual cisterna transitioning from early to late
compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)
In addition to retrograde transport from cis-Golgi to ER
(discussed below) the COPI coat is thought to mediate ret-
rograde transport within the Golgi complex to retrieve recy-
cling Golgi machinery to earlier compartments as Golgi
cisternae mature (Bonifacino and Glick 2004) In current
working models anterograde-directed COPI vesicles are tar-
geted to preceding Golgi compartments by the conserved
oligomeric Golgi (COG) complex a large multisubunit teth-
ering complex identi1047297ed through a combination of genetic
and biochemical approaches (Miller and Ungar 2012) COG
consists of eight subunits and belongs to the larger CATCHR
(complex associated with tethering containing helical rods)
family of tethering factors that includes the exocyst and
GARP complexes (Yu and Hughson 2010) In intra-Golgi
retrograde transport the COG complex appears to operate
as a tethering and fusion hub with multiple interactions that
link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE
proteins (Suvorova et al 2002) More speci1047297cally fusion
of retrograde-directed COPI vesicles with cis-Golgi mem-
branes is thought to depend on COG complex interactions
with a distinct SNARE complex consisting of Sed5 (Qa)
Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE
(Shestakova et al 2007) Mutations in COG complex subu-
nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this
stage there are no cell-free assays to measure COG-dependent
fusion of COPI vesicles to fully dissect underlying molecular
mechanisms (Miller and Ungar 2012)
Lipid requirements for Golgi transport
While the protein machinery underlying Golgi transport has
received much attention the role of speci1047297c lipid biosyn-
thetic and transfer pathways in Golgi traf 1047297cking remain
relatively understudied One of the 1047297rst connections for
a lipid requirement in transport through the Golgi complex
was the identi1047297cation and characterization of Sec14 as an
essential phosphatidylinositolphosphatidylcholine (PIPC)
transfer protein in yeast (Novick et al 1981 Bankaitis
et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-
ciated with Sec14 de1047297ciencies lead to an accumulation of
Golgi membranes and Golgi forms of secretory cargo Sec14
probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating
phospholipid homeostasis through presentation of PIs to
modifying activities such as the PI4 kinases (Schaaf et al
2008) Interestingly PI4P levels in the Golgi complex also
play a critical role in Golgi structure and function as dem-
onstrated by mutations in the essential PI4 kinase Pik1
which block transport through the Golgi (Walch-Solimena
and Novick 1999 Audhya et al 2000) More recently a di-
rect requirement for PI4P levels on Golgi organization has
been documented through characterization of the Golgi-
localized PI4P binding protein encoded by VPS74 (Schmitz
et al 2008 Tu et al 2008) Loss of Vps74 function results
in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind
to cytoplasmic sorting signals contained on Golgi resident
enzymes and to the COPI coat in addition to PI4P in sorting
Golgi-localized proteins into retrograde-directed vesicles In
this manner PI4P levels and Vps74 may function together
in dynamic recycling of Golgi modi1047297cation enzymes as cis-
terna containing nascent secretory cargo mature in accord
with Golgi maturation models Indeed the polarized dis-
tribution of PI4P across the Golgi with increasing concen-
trations from cis- to trans-compartments appears to play
several important roles in organization and transport through
the Golgi complex (Graham and Burd 2011)
The Return Journey Retrograde Traf1047297c viaCOPI Vesicles
Although it remains to this day somewhat controversial as to
the precise function (and thus direction) of COPI-mediated
vesicular traf 1047297c within the Golgi (Emr et al 2009) the role
of these vesicles in retrograde GolgindashER transport is well
established This is despite the original confusion in the 1047297eld
as to the directionality of COPI-mediated traf 1047297c yeast COPI
mutants generally have anterograde traf 1047297cking defects that
probably stem from indirect effects of blocking retrograde
transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component
Sec21 was identi1047297ed in the original sec mutant screen
(Novick et al 1980) advances in understanding this step of
the secretory pathway largely lagged behind and was informed
by the biochemical advances made in mammalian systems
(Sera1047297ni et al 1991) Once Sec21 was cloned and realized
to be an ortholog of the mammalian coatomer complex
(Hosobuchi et al 1992) biochemical analyses allowed the
identi1047297cation of all equivalent yeast subunits which were
398 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1728
in turn also subsequently identi1047297ed in a variety of genetic
screens as additional sec ret cop mutants (Duden et al
1994 Cosson et al 1996) The major advances in dissecting
the mechanisms of retrograde traf 1047297c have continued to be
led by biochemical approaches (Spang et al 1998 Spang
and Schekman 1998) with many recent high resolution
structures of the relevant coat (Lee and Goldberg 2010
Faini et al 2012 Yu et al 2012) and tether proteins (Ren
et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely
that the global structure of the yeast COPI coat is broadly
similar to that of mammals (Yip and Walz 2011) Indeed
current approaches make good use of yeast genetics ap-
proaches to test functional relevance of the structural data
yielding insight into areas including cargo selection (Michelsen
et al 2007) directionality of vesicle delivery (Kamena and
Spang 2004) and coattether in1047298uences on vesicle fusion
(Zink et al 2009)
Composition and structure of the COPI coat
Originally characterized from mammalian cells as a single
coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and
z-COP that correspond to the yeast proteins Cop1 Sec33
Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-
tively Although found as a large cytosolic complex it is now
appreciated that like the COPII coat COPI comprises two
separable layers an inner layer that functions in cargo bind-
ing composed of g- d- z- and b-COP and an outer layer
formed by a- b9- and e-COP (Figure 3) Furthermore sig-
ni1047297cant sequence homology was apparent between the inner
COPI coat and the adaptor subunits of the clathrin coat
system Indeed a recent structural analysis of the g z sub-
complex of the inner COPI coat shows clear homology with
the a s subunits of the AP2 clathrin adaptor with Arf1
bound at a site that corresponds spatially to the PI(45)P2
binding site on AP2 (Yu et al 2012) Although the structure
of the b d subcomplex remains to be determined homology
modeling suggests that it adopts a conformation very similar
to the b2ndash AP2 subunit and biochemical analyses suggest
that a second Arf1 molecule can bind to the PI(45)P2 bind-
ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat
which is most similar to the clathrin coat adaptors the outer
COPI coat shows homology with both clathrin and COPII
coats with b-propeller and a-solenoid domains forming
the building blocks of the putative cage Structural analysis
of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is
intermediate between that of the COPII and clathrin coats
the individual b-barrel and a-solenoid structures most
closely resemble the Sec13 Sec31 structure of the COPII
cage but they assemble in a clathrin-like triskelion (Lee
and Goldberg 2010) It remains unclear exactly how the
inner and outer layers come together either in solution
prior to assembly on the membrane or during vesicle forma-
tion although puri1047297ed yeast coatomer examined by single
particle electron microscopy suggests a somewhat 1047298exible
con1047297guration that would need to stabilize during poly-
merization or oligomerization on the surface of the mem-
brane (Yip and Walz 2011) This concept of structural
1047298exibility for the COPI coat is supported by recent EM anal-
ysis of COPI vesicles budded from synthetic liposomes
which showed striking structural diversity of coat arrange-
ment on the surface of the budded vesicles (Faini et al
2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-
malian proteins the yeast orthologs are highly likely to
adopt similar conformations Indeed the known structures
are consistent with the nonessential nature of Sec28 its
ortholog e-COP is a helical structure that interacts with
a-COPI but likely does not form part of the cage (Hsia and
Hoelz 2010 Lee and Goldberg 2010) probably rendering
it dispensable in vivo despite some destabilization of Cop1
(a-COP) in the sec28 mutant (Duden et al 1998)
Like the COPII coat COPI assembly on the membrane is
initiated by a small GTPase Arf1 which in addition to the N-
terminal amphipathic a-helix also contains a myristoyl
group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes
use of a common structural motif the Sec7 domain named
for the late Golgi GEF that is the target of the fungal me-
tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER
retrograde traf 1047297c two redundant GEFs Gea1 and Gea2
each with a Sec7 domain likely initiate coat assembly by
triggering local recruitment of Arf1 (Peyroche et al 1996
Spang et al 2001) Unlike the COPII system the GAP activ-
ity for the COPI coat is not an integral part of the coat itself
but is instead contributed by a separate protein known (not
surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash
GAP activity derives from two distinct proteins Gcs1 and
Glo3 with partially overlapping roles (Poon et al 1996
1999) Mammalian ArfGAP1 employs a lipid-packing sensor
domain to regulate its activity according to membrane cur-
vature becoming active on highly curved membranes likely
after vesicle formation has completed or at least progressed
enough as to permit Arf release without destabilizing the
coat (Bigay et al 2003 2005) Yeast Gcs1 also showed
a binding preference for conical lipids suggesting a similar
mechanism could regulate GTPase activity of the yeast COPI
coat (Antonny et al 1997b) However curvature-responsive
activity may not be the only mode of regulation of the COPI
GTPase cycle Coatomer itself also seems to in1047298uence Arf-
GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-
thermore the ability of some sorting signals on cargo pro-
teins to inhibit the coatomer-stimulated GAP activity directly
links coat recruitment to cargo selection (Springer et al
1999 Goldberg 2000) an appealing model whereby the
coat stably associates with the membrane only when bound
to cargo proteins (Springer et al 1999) Further complicat-
ing the problem is evidence that implicate ArfGAP proteins
as positive regulators of the COPI coat rather than negative
Early Events in Protein Secretion 399
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1828
regulators overexpression of any of the four yeast ArfGAPs
suppressed the lethality of an arf1 mutant (Zhang et al
1998 2003) Further yeast experiments also support an
active role for Gcs1 and Glo3 in cargo selection acting
on SNARE proteins prior to incorporation into vesicles to
promote Arf1 and coatomer interaction (Rein et al 2002
Schindler and Spang 2007 Schindler et al 2009) Clearly
the precise role of the GAP in the COPI system remains
to be fully understood complicated by con1047298
icting resultsfrom different labs andor systems and may in fact be mul-
tifaceted by serving both positive and negative roles at dif-
ferent stages during the vesicle formation process (Spang
et al 2010)
Cargo capture sorting signals cargo adaptorsand coat stimulators
Like other vesicle traf 1047297cking events retrieval of ER resident
proteins via COPI vesicles employs sorting signals most
notably the canonical retrieval motifs HDEL for soluble
lumenal cargoes and K(X)KXX for membrane proteins
(Figure 4) Soluble proteins bind to a retrieval receptor
Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-
criminate between similar but distinct motifs including the
canonical K(X)KXX which must be located at the C terminus
of the cargo and membrane-proximal to ensure ef 1047297cient
retrieval R-based motifs that only function when spaced
some distance from the membrane surface and other basic
motifs that remain to be fully dissected (Cosson et al
1998 Shikano and Li 2003) Yeast two-hybrid experi-
ments and subsequent mutagenesis analyses suggest that
the R-based motif binds at the interface between the b- and
d-COP subunits (Sec26 and Ret2 respectively) in a manner
that is distinct from KKXX binding to the coat (Michelsen
et al 2007) The site of KKXX recognition remains some-
what unclear Multiple lines of evidence support a role for
the a-b9-e-COP complex in KKXX binding (Cosson and
Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)
whereas direct cross-linking studies implicate the g-COP
subunit in KKXX binding (Harter et al 1996 Harter and
Wieland 1998)
In addition to retrieval motifs based on basic residues
diaromatic retrieval signals have also been identi1047297ed per-
haps best characterized for the p24 family of proteins albeit
largely using the mammalian family members (Strating
and Martens 2009) This class of signal likely binds to
the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor
platform to become receptive to additional cargo clients
(Beacutethune et al 2006 Strating and Martens 2009) Yet an-
other mode of cargo binding is represented by the SNARE
proteins that drive membrane fusion Unlike SNARE inter-
action with the COPII coat direct binding of SNARE sorting
signals with COPI components has not been observed In-
stead SNARE incorporation into COPI vesicles depends
on the activity of the Arf ndashGAP Glo3 although the precise
function of Glo3 in promoting a SNARE con1047297guration that
is favorable for vesicle capture remains to be fully dissected
(Rein et al 2002)
As with the COPII coat capture of cargo proteins into
retrograde COPI vesicles sometimes requires the action of
cargo adaptors The 1047297rst of these described was the HDEL
receptor Erd2 described above where the lumenal domain
likely provides ligand-binding function (Scheel and Pelham
1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al
1993) Another well-described cargo adaptor is the mem-
brane protein Rer1 (Nishikawa and Nakano 1993 Sato
et al 1995) which is important for the ef 1047297cient retrieval
and thus steady-state ER localization of some ER resident
proteins including the COPII GEF Sec12 and the translo-
con components Sec63 and Sec71 (Sato et al 1997) The
reason these proteins would require an escort back to the ER
rather than employing their own retrieval motifs is unclear
but Rer1 seems to bind these clients within their transmem-
brane domains via polar residues embedded within the hy-
drophobic environment (Sato et al 1996 2001) Sec12 and
Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had
no effect on Sec71 retrieval suggesting that Rer1 forms
a multivalent cargo receptor that has the capacity to bind
multiple cargo clients simultaneously (Sato et al 2003)
Yet another important player in COPI vesicle formation
is the class of proteins that seem to serve as coat nucleators
increasing or stabilizing the recruitment of the COPI coat
on the Golgi to stimulate retrograde traf 1047297c Although the
mechanistic details remain to be fully understood two
classes of protein seem to stimulate retrograde traf 1047297c by
modulating the ability of the COPI coat to form vesicles The
1047297rst description of this function was for a membrane protein
Mst27 which suppresses the lethality of a sec21-1 mutant
when overexpressed (Sandmann et al 2003) Mst27 and its
related binding partner Mst28 both bind to yeast coatomer
via KKXX motifs and this function is required for the sec21-1
suppression Although the endogenous function of Mst27
Mst28 is unclear the ability of these cargo proteins to stim-
ulate vesicle production was one of the 1047297rst concrete pieces
of evidence that cargo abundance can directly in1047298uence
vesicle format ion More recently a similar role has been
postulated for the abundant class of p24 proteins genetic
interactions between EMP24 and various COPI components
including SEC21 and the Arf ndashGAP GLO3 are suggestive
of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI
vesicles in vitro (Aguilera-Romero et al 2008) Since some
of the mammalian p24 proteins showed a capacity to mod-
ulate the GTPase activity of the COPI coat (Goldberg 2000)
it is tempting to link these observations by slowing the
GTPase activity of Arf1 the COPI coat might be stabilized
on the membrane prolonging the cargo-engagement step
and perhaps stimulating coat oligomerization to enhance
vesicle production
400 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 1928
Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion
Like other vesicle traf 1047297cking steps the 1047297nal stages of
delivery of COPI vesicles employ a long-distance tether to
bring the vesicle into proximity of the acceptor membrane
and SNARE proteins to drive membrane fusion (Spang
2012) The ER-localized tethering complex the Dsl1 com-
plex performs the tethering function recognizing COPI
vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs
prior to fusion (Figure 5) Originally identi1047297ed as a mutant
that was dependent on the presence of the dominant sly1-20
allele dsl1 mutants showed accumulation of vesicles at
restrictive temperature and were suppressed by overex-
pression of SEC21 although they also showed ER ndashGolgi
transport defects making a precise function dif 1047297cult to dis-
cern (VanRheenen et al 2001) Dsl1 forms a complex with
Dsl3 Sec39 and Tip20 to form the Dsl1 complex another
member of the CATCHR family of tethering complexes noted
for their extended helical rod structures (Lees et al 2010)
Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi
to the ER tip20 and dsl1 mutants showed genetic interac-
tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham
1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants
showed defects in fusion of COPI vesicles (Kamena and Spang
2004) the Dsl1 complex was localized to the ER (Kraynack
et al 2005) and Dsl1 interacts directly with multiple compo-
nents of the COPI coat (Andag and Schmitt 2003)
Recent structural analyses have generated an appealing
mechanistic model by which the extended Dsl1 complex
performs three functions by virtue of its ability to interact
with both the COPI coat and the fusogenic SNAREs (Ren
et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed
largely by Sec39 extends away from the ER membrane
with Dsl1 located at the membrane-distal end to ldquocatchrdquo
incoming COPI vesicles via an unstructured loop that would
interact directly with the coat via an a-helical structure
formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz
2010) Sec39 itself binds to the N-terminal domain of the ER
resident SNARE Use1 via a region that likely lies proximal
to the membrane (Tripathi et al 2009) and Tip20 contains
a second SNARE-binding site interacting with the N-terminal
domain of Sec20 (Ren et al 2009) In addition to bind-
ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion
maintaining individual SNAREs in an unpaired receptive
state and scaffolding assembly of the fusogenic SNARE
complex to promote fusion (Kraynack et al 2005 Ren
et al 2009) An additional role in vesicle uncoating is sug-
gested by the tendency of vesicles to accumulate en masse
under conditions of Dsl1 depletion (Zink et al 2009) COPI
shedding might be assisted by a Dsl1ndashCOPI interaction that
would prevent repolymerization of disassembled coat sub-
units or could be driven by conformational changes in the
Dsl1 complex that would capitalize on the ability of Dsl1 to
interact with both the outer a-e-COPI domain and a second
site on the inner d-COP subunit to prize the coat from the
membrane (Ren et al 2009 Zink et al 2009) Indeed neg-
ative stain EM images of the Dsl1 complex suggest a variety
of possible con1047297gurations although the mechanistic impact
of the different conformations with respect to coat and
SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that
may serve as a useful paradigm for other vesicle ldquotetheringrdquo
systems that may contribute to multiple layers of vesicle
uncoating docking and fusion in addition to their canonical
long-distance vesicle trapping function
Perspectives
Having moved from the ldquoparts listrdquo generated by numerous
genetic screens to molecular mechanisms de1047297ned by in vitro
assays where is the 1047297eld currently heading Emerging ques-
tions currently center on how the varied processes that drive
protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level
The biosynthesis of secretory proteins can be thought of as
a series of simple events (translationtranslocation post-
translational modi1047297cation chaperone binding forward
transport) but are these events more closely entwined than
we currently appreciate How are protein quality control
decisions made are they a simple outcome of a tug of war
between the ER-associated degradation machinery and the
forward transport machinery Adding a dominant ER export
signal to a misfolded protein could drive forward traf 1047297c
(Kincaid and Cooper 2007) but the converse experiment
of blocking ERAD of a different misfolded substrate did
not lead to its secretion (Pagant et al 2007) Understanding
the interplay between the folding degradation and export
machineries will be key in appreciating the intricate regula-
tion of secretory protein production and how the different
machineries might be coregulated to cope with the changing
secretory burden of the cell under different environmental
conditions
Additional questions stem from our relatively poor un-
derstanding of how the early secretory pathway is organized
and how this organization is maintained Although it is clear
that ER exit sites form discrete subdomains of the ER
(Rossanese et al 1999 Shindiapina and Barlowe 2010)
what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit
sites (Muniz et al 2001) driven by active processes or does
it re1047298ect the passive in1047298uence of speci1047297c lipid and protein
requirements for subsets of cargo molecules Similarly do all
secretory cargo proteins follow the same route through the
Golgi or are speci1047297c itineraries devised for distinct cargoes
that might also be driven by speci1047297c lipid microenvironments
andor post-translational modi1047297cation needs Larger-scale
questions also remain How is the cis-Golgi founded through
Early Events in Protein Secretion 401
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2028
homotypic fusion of COPII vesicles by heterotypic fusion of
COPII and COPI vesicles or by templating from an existing
cis-Golgi fragment that expands through delivery of COPII
and COPI vesicles Electron tomography of yeast cells show
distinct transport vesicles and Golgi cisternae but no apparent
intermediates (West et al 2011) How are vesicles targeted to
the correct destination Is there a role for the cytoskeleton in
vesicle delivery and how do COPI vesicles that bud from the
Golgi 1047297
nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different
transport events between different Golgi cisternae and do
tubular elements play a role in lipid and protein traf 1047297c as
they appear to do in mammalian cells Finally how are the
protein and lipid needs of the cell sensed and maintained to
ensure ef 1047297cient protein secretion which lies at the heart of
cell growth to permit cell division and how are the rates of
anterograde and retrograde traf 1047297c balanced to maintain the
correct morphology and distribution of the various secretory
organelles As in the past the facile genetics and accessible
biochemistry of the yeast system still hold promise in answer-
ing these questions with the development of new tools serv-
ing to strengthen the 1047297eld and provide new avenues forfurther exploration
Literature Cited
Aguilera-Romero A J Kaminska A Spang H Riezman and MMuniz 2008 The yeast p24 complex is required for the forma-tion of COPI retrograde transport vesicles from the Golgi appa-ratus J Cell Biol 180 713ndash720
Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734
Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160
Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072
Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684
Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851
Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537
Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424
Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689
Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344
Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359
Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416
Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc
Natl Acad Sci USA 83 9075ndash
9079Bankaitis V A D E Malehorn S D Emr and R Greene
1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281
Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108
Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879
Barlowe C L Orci T Yeung M Hosobuchi S Hamamoto et al1994 COPII a membrane coat formed by Sec proteins thatdrive vesicle budding from the endoplasmic reticulum Cell77 895ndash907
Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379
Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456
Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386
Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261
Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-
ticulum and Golgi complex J Biol Chem 276 43040ndash
43048Belden W J and C Barlowe 1996 Erv25p a component of
COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946
Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531
Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508
Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332
Beacutethune J M Kol J Hoffmann I Reckmann B Bruumlgger et al
2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021
Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756
Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277
Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645
402 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2128
Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924
Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566
Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253
Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042
Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166
Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124
Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182
Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363
Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during
endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460
Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cere- visiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400
Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060
Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789
Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-
erone machines Cell 92 351ndash
366Burda P and M Aebi 1999 The dolichol pathway of N-linked
glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity
of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552
Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944
Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213
Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66
Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165
Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155
Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373
Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200
Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun
et al 2010 Crystal structure of Get4-Get5 complex and its
interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970
Chartron J W C J Suloway M Zaslaver and W M Clemons Jr
2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132
Chen X C VanValkenburgh H Liang H Fang and N Green
2001 Signal peptidase and oligosaccharyltransferase interact
in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416
Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810
Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation
in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R
Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-
tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C
Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-
cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol
5 661ndash
667Connerly P L M Esaki E A Montegna D E Strongin S Levi
et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447
Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A
Miller 2012 ER cargo properties specify a requirement for COPII
coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-
lysine endoplasmic reticulum retention motifs Science 2631629ndash1631
Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798
Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870
Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431
Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206
Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404
Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931
Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802
Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885
drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport
vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal
surveillance complex that selects misfolded glycoproteins for
ER-associated degradation Cell 126 349ndash359
Early Events in Protein Secretion 403
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2228
Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645
Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805
Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-
subunit complex Nature 349 806ndash
808Doering T L and R Schekman 1996 GPI anchor attachment is
required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191
Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495
Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995
Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-
pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253
Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453
Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324
Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454
Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773
Fang H S Panzner C Mullins E Hartmann and N Green
1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465
Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158
Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054
Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89
Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion
complex SNARE proteins reclassi1047297
ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure
and organization of coat proteins in the COPII cage Cell 1291325ndash1336
Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840
Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296
Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399
Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cere- visiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306
Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol
112 27ndash
37Furgason M L C MacDonald S G Shanks S P Ryder N J
Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308
Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155
Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707
Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894
Gauss R K Kanehara P Carvalho D T Ng and M Aebi
2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793
Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802
Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759
Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741
Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane
protein related to giantin Mol Biol Cell 13 3761ndash
3774Gillingham A K A H Y Tong C Boone and S Munro
2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292
Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565
Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823
Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153
Goldberg J 1999 Structural and functional analysis of the ARF1-
ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through
a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions
by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization
of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218
Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion
404 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2328
into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604
Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304
Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol
Chem 285 21600ndash
21606Hann B C and P Walter 1991 The signal recognition particle in
S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-
uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533
Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406
Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535
Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD
integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521
Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654
Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906
Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579
Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101
Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase
a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850
Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049
Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329
Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567
Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction
map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene
required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605
Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276
Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691
Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233
Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430
Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem
277 49863ndash
49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al
2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500
Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33
Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411
Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697
Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77
Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733
Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY
Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289
Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R
Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830
Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441
Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463
Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471
Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628
Kraynack B A A Chan E Rosenthal M Essid B Umansky et al
2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977
Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595
Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027
Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in
Early Events in Protein Secretion 405
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2428
transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998
Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009
Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132
Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and
Golgi Annu Rev Cell Dev Biol 20 87ndash
123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al
2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617
Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297
Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196
Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207
Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483
Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478
Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186
Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006
Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283
21965ndash
21977Lussier M A M Sdicu F Bussereau M Jacquet and H Bussey
1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531
Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274
Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921
Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-
tional epitope Mol Cell 26 403ndash
414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564
Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708
Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275
Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709
Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010
McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159
Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-
log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164
Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849
Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217
Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113
Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509
Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726
Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897
Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115
Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756
Mossessova E L C Bickford and J Goldberg 2003 SNARE
selectivity of the COPII coat Cell 114 483ndash
495Mothes W S Prehn and T A Rapoport 1994 Systematic prob-
ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982
Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930
Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320
Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352
Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-
cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-
tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691
Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863
Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450
406 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2528
Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594
Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414
Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-
brane J Cell Biol 134 269ndash
278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like
protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892
Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183
Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070
Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274
Norgaard P V Westphal C Tachibana L Alsoe B Holst et al
2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562
Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862
Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215
Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469
Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563
Ogg S C W P Barz and P Walter 1998 A functional GTPase
domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354
Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420
Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805
Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011
Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993
Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413
Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570
Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198
Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655
Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528
Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481
Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415
Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420
Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807
Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated
by two ARF GAP proteins with overlapping function EMBO J18 555ndash564
Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077
Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222
Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891
Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol
Cell 3 789ndash
803Pucadyil T J and S L Schmid 2009 Conserved functions of
membrane active GTPases in coated vesicle formation Science325 1217ndash1220
Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669
Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404
Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129
Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-
derived transport vesicles J Cell Biol 114 219ndash
229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672
Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221
Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81
Early Events in Protein Secretion 407
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2628
Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780
Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628
Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in
yeast J Cell Biol 109 2641ndash
2652Rothman J E 1994 Mechanisms of intracellular protein trans-
port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization
of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065
Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476
Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503
Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442
Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217
Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365
Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113
Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767
Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208
Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA
96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog
Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532
Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174
Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477
Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-
dependent retrieval that requires the transmembrane domain
and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293
Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698
Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944
Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor
for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616
Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206
Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472
Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15
Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863
Schindler C F Rodriguez P P Poon R A Singer G C Johnston
et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375
Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988
Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647
Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534
Schuldiner M S Collins N Thompson V Denic A Bhamidipati
et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519
Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645
Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582
Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159
Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924
Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357
Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253
Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344
408 C K Barlowe and E A Miller
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2728
Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489
Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56
Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169
Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle
coat J Biol Chem 272 25413ndash
25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin
2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192
Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788
Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545
Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413
Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090
Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948
Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913
Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599
Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204
Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045
Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651
Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148
Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238
Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-
chemistry 41 11914ndash
11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101
Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159
Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537
Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in
the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142
Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307
Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509
Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477
Supek F D T Madden S Hamamoto L Orci and R Schekman
2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038
Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353
Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643
Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840
Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-
brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605
Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525
Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830
Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368
Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813
Travers K C Patil L Wodicka D Lockhart J Weissman et al
2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258
Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123
Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994
Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407
Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-
ochem 64 563ndash
591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44
VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742
VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231
Early Events in Protein Secretion 409
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis
7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway
httpslidepdfcomreaderfullsecretory-protein-biogenesis-and-traffic-in-the-early-secretory-pathway 2828
Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368
Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957
Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640
Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525
Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086
Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517
Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296
Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251
Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is
required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687
Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772
West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346
Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053
Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630
Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468
Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680
Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406
Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618
Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496
YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl
Acad Sci USA 88 517ndash
521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi
1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365
Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831
Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942
Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468
Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol
26 137ndash
156 Yu X M Breitman and J Goldberg 2012 A structure-based
mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542
Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796
Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330
Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419
Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link
between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416
Communicating editor T Davis