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Annu. Rev. Cell Dev. Biol. 2004. 20:87–123doi: 10.1146/annurev.cellbio.20.010403.105307

Copyright c© 2004 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on April 21, 2004

BI-DIRECTIONAL PROTEIN TRANSPORT BETWEEN

THE ER AND GOLGI

Marcus C.S. Lee,1∗ Elizabeth A. Miller,1∗Jonathan Goldberg,2 Lelio Orci,3 and Randy Schekman11Howard Hughes Medical Institute and Department of Molecular and Cell Biology,University of California, Berkeley, California; 2Howard Hughes Medical Institute andMemorial Sloan-Kettering Cancer Center, New York; 3Department of Morphology,University of Geneva Medical School, Switzerland; email: [email protected];e [email protected]; [email protected];[email protected]; [email protected]∗These authors contributed equally to this work

Key Words vesicle transport, COPI, COPII, coat proteins, cargo selection

■ Abstract The endoplasmic reticulum (ER) and the Golgi comprise the first twosteps in protein secretion. Vesicular carriers mediate a continuous flux of proteinsand lipids between these compartments, reflecting the transport of newly synthesizedproteins out of the ER and the retrieval of escaped ER residents and vesicle machinery.Anterograde and retrograde transport is mediated by distinct sets of cytosolic coatproteins, the COPII and COPI coats, respectively, which act on the membrane tocapture cargo proteins into nascent vesicles. We review the mechanisms that governcoat recruitment to the membrane, cargo capture into a transport vesicle, and accuratedelivery to the target organelle.

CONTENTS

INTRODUCTION TO THE SECRETORY PATHWAY . . . . . . . . . . . . . . . . . . . . . . . . 88GAINING TRANSPORT COMPETENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Folding and Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Accessing an Active Transport Zone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Bulk Flow Exit from the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Enrichment of Cargo Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

GENERATING A VESICLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Coat Recruitment and Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Cargo Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

DELIVERY OF VESICLES: MOTORS, TETHERING, AND FUSION . . . . . . . . . . 109Motor Proteins and Vesicle Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Vesicle Tethering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110SNARE Assembly and Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

NON-CANONICAL TRANSPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1081-0706/04/1115-0087$14.00 87

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88 LEE ET AL.

Anterograde Transport of Large Cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113COPI-Independent Retrograde Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

INTRODUCTION TO THE SECRETORY PATHWAY

Eukaryotic cells possess an elaborate endomembrane system that makes up thesecretory pathway (Figure 1). This network consists of a number of independentorganelles that function sequentially to effect protein secretion to the extracel-lular environment. Each compartment provides a specialized environment thatfacilitates the various stages in protein biogenesis, modification, sorting, and, ul-timately, secretion. The ER is the entry point into the secretory pathway for newlysynthesized proteins: Ribosomes dock onto a protein pore in the ER membrane,thereby releasing the nascent polypeptide into the lumen of the ER. The pri-mary role of the ER is to provide a milieu that facilitates protein folding. This isachieved by the presence of chaperones that massage a newly synthesized pro-tein into its correct conformation, sometimes through the catalysis of disulfidebond formation. Post-translational modification of nascent chains first occurs inthe ER, including addition of N-linked glycan chains and hydroxylation of prolineresidues.

The next station that a folded secretory protein visits is the Golgi apparatus, aseries of cisternae housing enzymes that function in glycan side chain modifica-tion and proteolytic cleavage. The modification of carbohydrate moieties can beextensive and proceeds sequentially, with distinct glycosyltranferases specificallylocated to cis-, medial-, or trans-compartments of the Golgi. The trans-Golgi net-work (TGN) serves as a sorting station that either sends proteins to the cell sur-face or diverts them to additional compartments of the endomembrane system,

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 1 Transport in the early secretory pathway. (a) Thin section electron micro-graph of an insulin-secreting cell. The ribosome-studded rough endoplasmic reticulum(ER) is visible, giving rise at one site to a budding profile (arrow). Such exit sites,also known as the transitional ER, produce numerous transport vesicles (TV) that willultimately be delivered to the stacked cisternae of the Golgi apparatus (Orci 1982).(b) Diagrammatic representation of ER-Golgi transport. COPII coat proteins medi-ate anterograde vesicle formation, selecting anterograde cargo and SNAREs. COPIIvesicles in plants and yeast fuse directly with the Golgi, but in mammalian cells theyseem to undergo homotypic fusion to generate a pleiomorphic structure known bothas the ER-Golgi intermediate compartment (ERGIC) and vesicular tubular clusters(VTC). The ERGIC/VTC is a site for concentrating retrograde cargo into COPI vesi-cles for delivery back to the ER. The ERGIC/VTC is delivered en bloc to the Golgi ina microtubule-dependent manner. Additional retrograde traffic from the Golgi properis also mediated by COPI vesicles.

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ER-GOLGI TRANSPORT 89

including the vacuole/lysosome, which is responsible for protein degradation, orto a variety of endosomal compartments that broadly function in protein sortingevents between the plasma membrane, TGN, and lysosome/vacuole.

Transport of proteins between these various compartments of the secretory path-way largely occurs via small vesicles that are generated at a donor compartment

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90 LEE ET AL.

and fuse with a downstream acceptor compartment. Cytoplasmic coat proteinssculpt vesicles by locally deforming the donor membrane. Distinct sets of coatsfunction at different steps in the secretory pathway: Clathrin and its various adap-tors function in a number of transport events between the TGN, endosome, lyso-some/vacuole, and plasma membrane; the COPI coatomer complex functions inretrograde transport between the Golgi and ER; and the COPII coat delivers pro-teins from the ER to the Golgi. In addition to their role in vesicle formation, coatproteins also drive the selective capture of proteins into vesicles by interacting withspecific signals present on the cytoplasmic domains of proteins. The combinationof coat recruitment to the correct donor membrane and signal-specific interactionsbetween the coat and cargo proteins contributes to the directionality and fidelityof vesicular transport. Further transport specificity is imparted by specific combi-nations of tethering complexes and fusion assemblies known as SNAREs (solubleN-ethylmaleimide-sensitive factor attachment protein receptors), both of whichfacilitate fusion of vesicles with acceptor organelles.

This review focuses on the mechanisms of vesicular transport between the firsttwo compartments of the secretory pathway, the ER and the Golgi. Transportbetween these organelles occurs in two directions: anterograde (ER-Golgi) trans-port delivers newly synthesized cargo proteins to the Golgi, whereas retrograde(Golgi-ER) transport retrieves ER residents and other machinery that constantlycycle between these two compartments (Figure 1). We consider the requirementsfor access of cargo proteins to nascent vesicles, as well as the mechanisms ofvesicle formation, with particular emphasis on the detailed molecular interactionsdriving this process that have been elucidated through recent structural analysis ofthe various coat proteins.

GAINING TRANSPORT COMPETENCE

An important feature of vesicular transport is that, for the most part, only designatedcargo proteins are packaged into a nascent vesicle; organelle resident proteins failto be incorporated or are included at their prevailing concentration (Malkus et al.2002). Designated cargo proteins need not only refer to newly made biosyntheticcargo; a variety of cellular machinery proteins that facilitate various aspects ofvesicle formation and downstream fusion are also specifically recruited. In anycase, a number of factors govern whether a bona fide cargo protein will be rec-ognized and subsequently captured into a newly forming vesicle. Some of thesefactors are common to both anterograde and retrograde pathways, whereas othersare more specific.

Folding and Assembly

Newly synthesized proteins entering the anterograde COPII pathway must escapethe clutches of the ER quality control system, which monitors the folding status of

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proteins and targets for degradation those that are terminally misfolded. The pre-cise mechanisms that mediate this assembly checkpoint remain poorly understood,but the process is thought to be important in preventing “proteotoxicity” associatedwith aberrant secretion of improperly assembled proteins (Kim & Arvan 1998).The observation that misfolded proteins such as a temperature-sensitive mutantof the vesicular stomatitis virus G protein (ts045 VSVG) show prolonged interac-tions with ER chaperones led to the suggestion that extensive association with animmobile matrix of resident proteins would preclude incorporation of the aberrantcargo protein into a COPII vesicle (Hammond & Helenius 1994). Fluorescencemicroscopy of a green fluorescent protein (GFP)-tagged version of ts045 VSVGrevealed that the mobility of the misfolded protein within the ER was identicalto that observed for the correctly folded form, suggesting that the mechanism ofretention is not immobilization (Nehls et al. 2000). However, depletion of ATP inthese cells caused a decrease in both the mobile fraction of ts045 VSVG and thediffusion rate of a soluble ER marker. Hence, under conditions where chaperone-substrate dynamics are perturbed, the ER matrix may be more viscous and therebyimpede the mobility of a misfolded substrate. How these dynamics might influencethe availability of a cargo protein for packaging into a vesicle is unclear; however,the mobility of a substrate protein within the ER may regulate access to a privilegedsite of COPII budding (see below).

It seems likely that the vast majority of cargo proteins that transit the retrogradepathway in COPI vesicles are by nature fully folded; this pathway functions toretrieve escaped ER residents and to recycle the machinery that mediates vesicleformation and fusion. However, recent studies examining the fate of misfoldedsoluble proteins have indicated that some misfolded proteins travel to the Golgibefore being retrieved to undergo ER-associated degradation (ERAD) (Caldwellet al. 2001, Taxis et al. 2002, Vashist et al. 2001). In this respect, the inverse of afolding requirement is likely to act in recruitment of these cargoes into COPI vesi-cles; only misfolded proteins will gain access to a vesicle and thereby be preventedfrom further transport through the secretory pathway. Presumably, this recruitmentacts via an unknown transmembrane receptor that couples the misfolded substrateto the cytoplasmic coat.

Accessing an Active Transport Zone

In most eukaryotes, the endoplasmic reticulum is not a homogeneous environ-ment. Fluorescence and immunoelectron microscopy of COPII proteins reveals arestricted localization to domains of the ER that have been variously designated asER exit sites (ERES) or transitional ER (tER), which represent regions dedicated togenerating COPII vesicles (Kuge et al. 1994, Orci et al. 1991, Pagano et al. 1999).Precisely how these distinct zones are maintained is unclear, and the repertoire ofproteins that mark these sites is not fully characterized. Furthermore, the functionalsignificance of these privileged budding sites is not known, although the degree oforganization of these ER zones may influence the morphology of newly forming

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92 LEE ET AL.

Golgi elements (Rossanese et al. 1999). An additional role for these sites in regu-lating the quality control of cargo selection is implicated by immunofluorescencemicroscopy examination of the colocalization of ts045 VSVG with COPII pro-teins under permissive and restrictive conditions (Mezzacasa & Helenius 2002).When ts045 VSVG is misfolded, it fails to gain access to the ERES, which arealso devoid of ER resident chaperones. Upon shift to the permissive temperature,ts045 VSVG rapidly folds and becomes associated with ERES, suggesting thatthe folding status of this cargo protein is monitored such that only the fully foldedprotein gains access to a privileged site of COPII vesicle formation.

The localization of COPI proteins on the Golgi apparatus does not appear toshow the same degree of organization. If the main function of these specializedzones in the anterograde pathway is to act in quality control, this apparent lackof organization in the retrograde pathway could merely reflect the absence of aspecific checkpoint that cargo proteins must pass. However, in mammalian cellsthe selective and efficient capture of cargo proteins into COPI vesicles may makeuse of an additional membrane-bound compartment, the ER-Golgi intermediatecompartment (ERGIC), also known as vesicular tubular clusters (VTC). This com-partment is thought to arise from the homotypic fusion of COPII vesicles but isalso marked by COPI proteins. Immunogold electron microscopy of cargo andcoat proteins suggests that the ERGIC is a main sorting station for the retrieval ofescaped ER resident proteins (Martinez-Menarguez et al. 1999) and therefore mayrepresent a functionally distinct budding zone similar to that of the tER/ERES inthe anterograde pathway.

Bulk Flow Exit from the ER

Once proteins destined to leave the ER are correctly folded and have escaped theretention mechanisms described above, they may enter transport vesicles eitherat their prevailing concentrations (bulk flow) or at concentrations significantlyhigher than that in the general ER. Early support for rapid bulk flow transportof soluble cargo was obtained by measuring the rate of secretion of a glycosy-lated acyltripeptide from mammalian cells (Wieland et al. 1987). In a recent study,however, a comparison of three separate bulk flow markers (a glycosylated acyl-tripeptide, ER-lumenal GFP, and total phospholipid) revealed that only ∼0.6–2%of each was captured into COPII vesicles generated from a cell-free reaction usingyeast microsomes (Malkus et al. 2002). Thus passive sampling of the lumenalcontents of the ER appears to be an inefficient mode of transport, but one thatis nonetheless used by some secreted proteins. Exocrine pancreatic cells secretethe soluble enzymes amylase and chymotrypsinogen, which are concentrated dur-ing passage through the secretory pathway and accumulate in secretory granulesprior to regulated exocytosis. Immunoelectron microscopy analysis of amylase andchymotrypsinogen in the early secretory pathway demonstrated that enrichmentdid not occur in COPII-coated ER buds but in the VTC, where they were con-centrated 1.5-fold and 16-fold, respectively (Martinez-Menarguez et al. 1999).

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Enrichment of these proteins in the VTC most likely reflects exclusion from retro-grade COPI vesicles rather than selective capture into anterograde COPII vesicles(Martinez-Menarguez et al. 1999). It remains to be seen whether this process ofconcentration by exclusion is used more widely or is limited to especially abundantproteins or proteins that do not require rapid delivery to their final destination.

Enrichment of Cargo Proteins

Specific enrichment of both membrane and soluble cargo proteins in transportvesicles occurs at concentrations ∼3- to 50-fold higher than the bulk flow markersdescribed above. This enrichment is achieved by interaction of the cytoplasmiccoat with distinct sorting signals on cytoplasmic segments of membrane cargoproteins. To be recognized by the coat machinery, these signals must be acces-sible and in an appropriate conformation that may be influenced by a number offactors, including the folding status of the protein (discussed above), interactionswith accessory proteins or transport receptors, or an oligomerization state thatmay influence the presentation of positive sorting signals or masking of retentionsignals.

TRANSPORT RECEPTORS Soluble cargo proteins and GPI-anchored membrane pro-teins that do not present cytoplasmic signals must instead interact with specifictransmembrane receptors that serve to link these lumenal substrates to the cyto-plasmic coat. The yeast membrane protein Erv29 forms a complex with a precursorof the soluble pheromone, α-factor, and is required for the efficient packaging ofpro-α-factor into a COPII vesicle (Belden & Barlowe 2001b). Deletion of Erv29results in a reduction of the α-factor precursor packaging to levels similar to bulkflow markers (Belden & Barlowe 2001b, Malkus et al. 2002). In addition to α-factor, Erv29 is also likely to package other soluble proteins, including the vacuolarhydrolases, carboxypeptidase Y, and proteinase A; however, other secretory pro-teins such as invertase are packaged normally in Erv29-depleted cells, suggestingadditional transport receptors remain to be identified (Caldwell et al. 2001). Inmammalian cells, a lectin, ERGIC-53, is required for the secretion of a number ofglycoproteins, including the clotting proteins factor V and factor VIII (Appenzelleret al. 1999). Mutations in either ERGIC-53 (also called LMAN1) or an associatedprotein, MCFD2, cause an autosomal recessive bleeding disorder as a result of acombined deficiency of the clotting factors (Zhang et al. 2003). Although not wellcharacterized, the interactions between soluble cargo proteins and transmembranesorting receptors are likely to be influenced by the folding state of the cargo pro-tein, providing an additional level of quality control. In the case of ERGIC-53,this quality control function may be facilitated by sequential interaction of thecargo substrate with the lectin chaperones calnexin/calreticulin and ERGIC-53.Furthermore, the mechanism by which these transport receptors release their cargoupon deposition in the Golgi remains to be determined, although by analogywith other transport steps in the secretory pathway, a change in pH or another

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physiologic condition may cause dissociation of the cargo and its receptor andthereby allow retrieval and reuse of the receptor.

In the retrograde pathway, a different transmembrane protein fulfills the functionof transport receptor. Soluble ER resident proteins bear a specific retrieval signal(KDEL in mammals, HDEL in yeast) that mediates interaction with the KDELreceptor (Erd2 in yeast). The KDEL receptor itself possesses a non-canonicalcytoplasmic di-lysine retrieval motif that contributes to interaction with the COPIcoat in conjunction with a phosphoserine residue that is also important for ER-Golgi retrieval (Cabrera et al. 2003). Although the precise mechanisms by whichthe KDEL receptor binds its ligands are not known, a number of point mutations onthe lumenal surface of the receptor abrogate binding to KDEL peptides, implicatingthese residues as a ligand-binding pocket (Scheel & Pelham 1998). Furthermore,ligand binding to the KDEL receptor is thought to trigger uptake of the assembledcomplex into COPI vesicles (Lewis & Pelham 1992). This regulated transportmay be driven by the ligand-induced oligomerization of the receptor, which inturn may stimulate interaction with the COPI coat (Majoul et al. 2001). In vitrobinding assays have demonstrated that the affinity of the KDEL receptor for itsligands shows a striking pH dependency, suggesting that subtle differences in theluminal environment will allow release of KDEL-containing proteins upon fusionwith the ER (Wilson et al. 1993). Thus many aspects of the KDEL retrieval pathwaymirror similar ligand-induced endocytic events at the plasma membrane: ligand-induced dimerization, specific recruitment to a budding site, and ligand releaseupon a change in luminal pH.

Similar to soluble cargoes, GPI-anchored proteins project no signals into thecytoplasm, suggesting a requirement for a transmembrane receptor. Yeast Emp24may function as the receptor for the GPI-anchored membrane protein Gas1; Emp24can be cross-linked to Gas1 in transport vesicles, and packaging of Gas1 intotransport vesicles is reduced in Emp24-depleted cells (Muniz et al. 2000). A furtherspecialized role for Emp24 in ER export is suggested by the observation that Gas1and other GPI-anchored proteins enter transport vesicles that are distinct from thosethat carry other cargo proteins such as α-factor and amino acid permeases (Munizet al. 2001). Yet another role for the p24 family of proteins (of which Emp24 is amember) in the fidelity of protein sorting has been implicated by the observationthat ER resident proteins are secreted from cells harboring deletions in p24 proteins(Elrod-Erickson & Kaiser 1996). Likewise, mutation of a Caenorhabditis elegansp24 protein, sel-9, causes the secretion of an aberrantly folded plasma membranereceptor that is normally retained in the ER (Wen & Greenwald 1999). Variousmodels explaining the mechanism of p24 function in sorting fidelity remain to befully tested (Kaiser 2000), and the possibility remains that this family of proteinshas multiple functions, perhaps in both anterograde and retrograde traffic.

PACKAGING CHAPERONES In some cases, cargo receptors also function as molec-ular chaperones. The Drosophila protein, NinaA, likely functions as a peptidyl-prolyl cis-trans isomerase and is required for proper ER export of rhodopsin.

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NinaA is also associated with rhodopsin in secretory vesicles and is thought to actboth as chaperone and escort, accompanying rhodopsin in its journey through thesecretory pathway (Baker et al. 1994, Colley et al. 1991). However, a direct rolefor NinaA as a cargo receptor for uptake into ER-derived vesicles has not yet beendemonstrated. Whereas NinaA seems to accompany its substrate out of the ER,another putative chaperone, yeast Shr3, seems to facilitate packaging of its sub-strates yet is retained in the ER (Kuehn et al. 1996). Cells lacking Shr3 accumulateamino acid permeases in the ER. Shr3 interacts with the COPII coat and throughthis interaction facilitates packaging of the permeases without being captured intoa vesicle itself (Gilstring et al. 1999). Additional examples of membrane proteinsthat seem to facilitate ER export of transmembrane substrates include yeast Chs7,the mutation of which causes ER accumulation of a plasma membrane chitinsynthase (Trilla et al. 1999); yeast Erv14, which accompanies Axl2 out of theER (Powers & Barlowe 1998); yeast Pho86, deletion of which causes the phos-phate transporter Pho84 to accumulate in the ER (Lau et al. 2000); and C. elegansodr-4, which is involved in secretion of odorant receptors (Dwyer et al. 1998).Exactly how these packaging chaperones function in ER export remains to becharacterized.

The requirement for accessory factors to mediate transport of transmembraneproteins is common to both the anterograde and retrograde pathways. The yeastGolgi membrane protein, Rer1, facilitates retrieval of a number of escaped ERresident membrane proteins, including Sec12 and Sec71 (Sato et al. 1996, 1997).Interactions between Rer1 and its substrates within transmembrane domains gov-ern retrieval, although separate regions in Rer1 seem to interact with Sec12 andSec71 (Sato et al. 2003). Whether Rer1 itself is taken up into COPI vesicles re-mains to be determined; however, it interacts directly with subunits of the COPIcoat and contains an essential cytoplasmic signal that resembles the canonicalCOPI di-lysine retrieval motif, as well as a separate tyrosine signal (Sato et al.2003). The mechanism by which Rer1 releases its ligands upon deposition in theER remains to be fully characterized. However, the observation that many of thesubstrates that use Rer1-mediated retrieval are oligomeric proteins suggests thatRer1p functions to retrieve escaped monomers that have a higher affinity for theirER-localized partners. Retrieval to the ER should result in a displacement of Rer1as the cargo monomer is restored to an oligomeric form.

OLIGOMERIC ASSEMBLY Instead of using accessory proteins to facilitate transport,many cargo molecules rely on assembly cues to drive uptake into a transport vesi-cle. This seems to be true for both biosynthetic cargo and the transport machinerythat cycles between the ER and Golgi. The mammalian lectin ERGIC-53 con-tains a cytoplasmic sorting signal that directs interaction with the COPII coat.However, homo-oligomerization of ERGIC-53 is a prerequisite for efficient ERexport: Mutation of cysteine residues that participate in intermolecular disulfidebonds disrupts ER-Golgi transport (Nufer et al. 2003). Similar oligomerization-dependent ER export has been reported for the yeast homologs of ERGIC-53,

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Emp46, and Emp47, where disruption of coiled-coil domains that mediate assem-bly impairs uptake of these proteins into COPII vesicles but has little effect oncargo-coat interactions (Sato & Nakano 2003). Although the mechanism by whicholigomerization might affect ER export remains unclear, one could imagine thatpresenting an oligomeric signal might dramatically increase the affinity of the coatfor a specific cargo protein, thereby enriching the assembled complex in a nascentbudding zone. Alternatively, the coat may not recognize a signal unless it is pre-sented in a specific conformation that is achieved only through oligomerization.Such a contextual arrangement of ER export signals has been proposed for theyeast ER/Golgi proteins Erv41/Erv46 (Otte & Barlowe 2002).

Yet another requirement for oligomerization may result from the absence of ERexport signals on specific partners within a heteromeric complex. For example, asubfamily of mammalian inwardly rectifying potassium channels, Kir3, possessesfour members that can combine in different permutations to yield active channels.Homotetramers of one member, Kir3.1, are not functional because these assembliesare retained in the ER. Kir3.1 lacks any ER export signals and therefore relies onsignals found on its partners for efficient transport from the ER (Ma et al. 2002).Such reliance on heteromeric assembly may reflect an additional layer of qualitycontrol, such that only properly assembled complexes are selected for forwardtransport, leaving unassembled monomers behind to search for an appropriatepartner or to interact further with the ER folding machinery.

Similar assembly-dependent transport has been described in retrograde re-trieval, although this represents an inverse assembly requirement such that fullyassembled complexes are not a substrate for COPI retrieval. Individual subunits ofa number of oligomeric cell surface receptors and channels possess ER retrievalmotifs that are masked when the holocomplex is correctly assembled (Letourneuret al. 1995, Mallabiabarrena et al. 1992, Margeta-Mitrovic et al. 2000). This mask-ing of retention signals is presumed to be a steric effect of the interaction of adjacentcytoplasmic domains to prevent access to the COPI coat. In contrast to intersubunitmasking of retrieval motifs, recent studies on the secretion of oligomeric potas-sium channels implicate an external component in regulating forward transport:14-3-3 proteins. These potassium channels possess di-arginine motifs that func-tion largely as retrieval signals, mediating COPI-dependent retrieval to the ER.Correct assembly into an oligomeric structure obstructs this ER retrieval motif byrecruiting specific members of the 14-3-3 protein family (O’Kelly et al. 2002, Yuanet al. 2003). Binding of 14-3-3 thus competes with the COPI coat for interactionwith these substrates, preventing uptake into the retrograde pathway. Again, thefunction of this assembly-mediated retrieval seems to be in denying access to thelater secretory pathway for proteins that are improperly assembled and therebygiving these complexes a second chance for interaction with the folding apparatusof the ER.

A common theme in this discussion of the requirements for specific cargo cap-ture into budding vesicles is that the process seems tightly linked to quality control.Whether through the use of specific cargo receptors that might monitor the folding

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state of a protein or the correct presentation of oligomeric signals or masking ofretrieval motifs that indicates correct assembly, cells have multiple checkpointsthat ensure only legitimate cargo substrates are delivered to the appropriate com-partment. Furthermore, the retrograde retrieval pathway provides an extra layer ofquality security in giving inappropriately assembled substrates a second chance atachieving the correct conformation. Our next challenge lies in understanding howthese different checkpoints are mechanistically linked to the process of vesiclebiogenesis.

GENERATING A VESICLE

The process of constructing a vesicle follows a relatively prescribed course of ac-tion that is common to all vesicle budding events (Figure 2). The most fundamentalof these is the localized curvature of the membrane that sculpts a vesicle out of thedonor compartment. Generally, this curvature is driven by the coat subunits thatdefine the vesicle type or by additional effectors that may locally deform a mem-brane at specific sites. To this end, coat components must be specifically recruitedto appropriate sites of vesicle budding and interact there with bona fide cargo pro-teins to generate a functional vesicle. Generally, small GTP-binding proteins thatact as molecular switches to turn on coat assembly drive the initial recruitment ofthe coat. Additional coat components are subsequently recruited, cargo moleculesare recognized, and coat polymerization drives release of the nascent vesicle fromthe donor membrane. Our understanding of the molecular mechanisms that drivethese processes has been greatly enriched by the structural characterization of indi-vidual coat components. Although many of the structural details of the anterogradeand retrograde coat complexes are remarkably distinct, these two pathways sharemany broad similarities.

Coat Recruitment and Assembly

ACTIVATION OF THE SMALL G PROTEINS Coat assembly begins through activa-tion of the small G proteins; Sar1 for COPII and Arf1 for COPI (Figure 3). Bothproteins cycle between cytosolic and membrane-associated pools, with GTP bind-ing generating the active, membrane-bound state. As with other small G proteinssuch as Ras and Rho, exchange of GDP for GTP is catalyzed by guanine nucleotideexchange factors (GEFs), but Sar1 and Arf1 are unusual in that the exchange re-action can proceed only in the presence of a membrane surface (Antonny et al.2001, Paris et al. 1997). This requirement for membranes upon GTP binding re-sults from the exposure of an N-terminal amphipathic α-helix that is unique to theARF family. In Arf1 this helical domain extends for 17 residues and is capped atthe N terminus by a myristoyl group (Antonny et al. 1997), whereas Sar1 is notlipid modified but has a longer domain of approximately 23 residues (Huang et al.2001). In the GDP state the helix is retracted into a surface pocket, and truncation

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98 LEE ET AL.

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of this helical region permits nucleotide exchange to proceed in the absence ofmembranes (Antonny et al. 1997, Bi et al. 2002).

Structural information now available for both the GDP- and GTP-bound statesof Sar1 and Arf1 illustrate how GTP binding induces conformational changes thatcouple membrane binding with coat recruitment (Amor et al. 1994, Bi et al. 2002,Goldberg 1998, Huang et al. 2001). As with other Ras-like G proteins, the classicalswitch I and switch II regions are rearranged to accommodate the γ phosphate ofGTP (Bi et al. 2002, Goldberg 1998). Uniquely to the ARF family, however, thisrearrangement is coupled to a 7 A displacement of a β-hairpin, consisting of theβ2–β3 strands, which connect the switch regions, into the pocket occupied bythe N-terminal helix (Bi et al. 2002, Goldberg 1998). Thus insertion of the N-terminal helix into the membrane bilayer is a prerequisite for GTP exchange, andretraction of this membrane anchor can occur only upon GTP hydrolysis (Figure3). This requirement for membranes coupled with localized GEF activity may helpto restrict coat recruitment to the appropriate compartment.

Despite similarities in the conformational changes adopted by Sar1 and Arf1upon GTP binding, the exchange factors that act on each protein appear to beunrelated and are likely to catalyze nucleotide exchange by different mechanisms.Sec12, the GEF for Sar1, is a type II membrane protein located in the ER (Barlowe& Schekman 1993). In Saccharomyces cerevisiae, Sec12 appears to be distributedthroughout the ER. However, in the related budding yeast Pichia pastoris, Sec12 isfound in punctate spots that colocalize with other COPII subunits, suggesting Sec12restricts COPII recruitment to distinct transitional ER budding zones (Rossanese

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2 Coat assembly and vesicle budding. (a) COPII coat assembly is initiatedby the ER resident, Sec12, which serves as a guanine nucleotide exchange factor(GEF) for the small GTPase, Sar1 (1). GTP binding by Sar1 exposes an amphipathicα-helix that facilitates association with the ER membrane. Membrane-associated Sar1recruits the Sec23-24 heterodimer (2), and this complex interacts with cargo proteinsvia specific sorting signals (3). The Sar1-Sec23-Sec24 complex then recruits the Sec13-31 heterotetramer (4), which is thought to polymerize the coat and drive membranedeformation to yield a COPII vesicle, shown in thin section (Schekman & Orci 1996).(b) COPI coat assembly is similar in that coat recruitment is initiated by GDP-GTPexchange on Arf1, mediated by the ARF GEF, Gea1 (1). Membrane-bound Arf1 thenrecruits the preassembled coatomer complex, which contains seven subunits: the α/β ′/εcomplex and the β/γ /δ/ζ complex (2). The COPI coatomer complex likely containsmultiple cargo recognition sites on separate subunits that mediate recruitment of cargoproteins (3). Ultimately, the coat is polymerized by an unknown mechanism, andmembrane curvature may facilitate the recruitment of an Arf GTPase-activating protein(ARF GAP), stimulating GTP hydrolysis by Arf and subsequent dissociation from themembrane (4). A purified COPI vesicle is shown in thin section (Schekman & Orci1996).

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100 LEE ET AL.

Figure 3 Nucleotide-dependent conformational changes in Arf1. A comparison ofthe structures of Arf1 bound to GDP or a nonhydrolyzable analog of GTP shown inidentical orientation. The amphipathic N-terminal helix of Arf1 (red) is sequestered ina hydrophobic groove in the GDP-bound state. Exchange of GDP for GTP is associatedwith the insertion of this N-terminal helix into the membrane bilayer and the loss ofthe hydrophobic binding pocket. Subsequent retraction of the helix is contingent onGTP hydrolysis.

et al. 1999). The cytosolic domain of Sec12 is predicted to form WD40 repeats,folding as a seven-bladed β-propeller (Chardin & Callebaut 2002). In this respectit is reminiscent of the GEF for Ran, RCC1, which also forms a seven-bladedβ-propeller (Renault et al. 2001).

In contrast to Sar1, several different exchange factors exist for Arf1, likelyreflecting the role of Arf1 in multiple vesicle budding events. Although widelydifferent in size, all Arf GEFs contain a catalytic domain of ∼200 amino acids,known as the Sec7 domain, and thus are likely to mediate nucleotide exchangeby the same mechanism (Donaldson & Jackson 2000). Structural and biochemicalanalyses of the Sec7 domain reveal an extensive interaction with the switch I andII regions of Arf1, positioning a catalytic glutamine residue on the Sec7 domaininto the GTPase active site. Displacement of the bound nucleotide occurs throughdisruption of the nucleotide β-phosphate group and the Mg2+ ion (Goldberg 1998,Mossessova et al. 1998). The complex between the Sec7 domain and Arf1-GDP isthe target of the fungal metabolite Brefeldin A, which is commonly used to disruptArf1-dependent trafficking (Mossessova et al. 2003b, Robineau et al. 2000).

ARF GEFs fall broadly into two classes. The large GEFs such as Gea1, Gea2,and Sec7 in yeast, and GBF1 and BIG1 in humans, are all larger than 100 kD. The

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small GEFs (<100 kD), such as ARNO and cytohesin-1, do not have homologsin yeast (Jackson & Casanova 2000). The Gea1 and Gea2 proteins, which haveoverlapping functions, are largely responsible for stimulating the formation ofretrograde COPI vesicles in yeast, whereas Sec7 is located mainly at the TGN(Peyroche et al. 1996, Spang et al. 2001). The mammalian ortholog of Gea1/2,GBF1, is located at the cis-Golgi and VTCs and is likely to perform the equivalentfunction (Zhao et al. 2002).

Unlike Sec12, the exchange factor for Sar1, all ARF GEFs are cytosolic proteins.How then is Arf1 activation directed to the appropriate membrane? One likelyexplanation is that additional lipid- and protein-binding domains could localizedifferent GEFs to their target membranes. Recently Gea1/2 recruitment to theGolgi membrane was found to be stimulated by interaction with a transmembraneprotein, Gmh1 (Garcia-Mata et al. 2003). In addition, small GEFs such as ARNOhave a pleckstrin homology domain that mediates binding to phosphoinositides,although specific lipid-binding domains on the larger GEFs have yet to be identified(Jackson & Casanova 2000). In addition, recruitment of Arf-GDP to the Golgi bythe cytosolic tails of some p24 cargo proteins suggests a second mechanism torestrict Arf1 activation (Gommel et al. 2001).

COAT ASSEMBLY ON THE MEMBRANE Membrane-bound Sar1-GTP and Arf1-GTPare now primed to recruit additional coat proteins. The COPI coat consists of sevensubunits, a subset of which appears to be structurally related to the adaptor protein(AP) complex found in clathrin-coated vesicles. The COPII coat has four subunitsthat bear no apparent relation to either the COPI or AP coat complexes. Both theCOPI and COPII coat proteins are conserved between yeast, plants, and mammals.Despite the lack of similarity between the COPI and COPII subunits, the commonfunction of both coats is to promote membrane curvature and cargo protein capture.

COPII coat recruitment At the ER, Sar1-GTP sequentially recruits two cytoso-lic complexes, the Sec23-Sec24 heterodimer and the Sec13-Sec31 heterotetramer(Figure 2). On synthetic liposomes, these five components are sufficient to deformthe membrane and generate coated vesicles (Matsuoka et al. 1998). Sec23 is theGTPase-activating protein (GAP) for Sar1, and binds to Sar1-GTP via the switchregions, on the side opposite to the membrane anchor (Bi et al. 2002, Yoshihisa et al.1993). The structure of the Sar1-Sec23 complex indicates Sec23 may stimulate theintrinsically slow GTPase activity of Sar1 by supplying an “arginine finger” intothe catalytic site (Bi et al. 2002). Sec24 is responsible for the majority of the cargorecognition decisions (see below). Despite low sequence identity (<14%), Sec23and Sec24 are structurally similar and form a bowtie-shaped complex approxi-mately 15 nm in length (Figure 4). A concave inner face enriched in basic residuesmay act to enhance membrane association and facilitate membrane curvature (Biet al. 2002).

Sec13-31 forms the outer layer of the coat and cannot be cross-linked to thelipid membrane, unlike the other subunits (Matsuoka et al. 2001). Both Sec13

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102 LEE ET AL.

Figure 4 Structure of the Sar1-Sec23-Sec24 complex. Sar1 is shown in red, Sec23in yellow and Sec24 in green. The three panels illustrate successive 90◦ rotations ofthe complex, with the central panel showing the predicted membrane-proximal surfacefacing forward. This concave inner face is shown in relation to the curved membrane ofa 60-nm vesicle (gray line, left and right panels). On Sec24, peptides encompassing ERexport signals are shown bound to the A-site (pink) and B-site (blue), and the criticalarginine residue in the C-site is highlighted in red.

(∼33 kD) and Sec31 (∼140 kD) are predicted to contain WD40 repeats, and astable complex of two molecules each of Sec13 and Sec31 can be isolated fromyeast cytosol (Lederkremer et al. 2001). Electron microscopy images show thatSec13-Sec31 forms an elongated molecule ∼30 nm long with multiple globulardomains (Lederkremer et al. 2001, Matsuoka et al. 2001). Although the exactnature of the interaction between Sec13-31 and Sec23-24 is not known, Sec13-31 is likely to function as a structural scaffold to cross-link adjacent Sec23-24complexes, forming a coat lattice that propagates membrane curvature.

Prior to Sec13-Sec31 recruitment, the lifetime of the Sar1-Sec23-24 prebuddingcomplex (∼30 s on synthetic liposomes) is likely to be sufficiently long as to allowlateral diffusion on the ER membrane and cargo capture by Sec24. Subsequentbinding of Sec13-31 accelerates the Sec23-mediated GAP activity by an orderof magnitude (Antonny et al. 2001). Thus the internal timer for Sar1 release byGTP hydrolysis is controlled by the stepwise assembly of the COPII coat proteins.This sequential assembly is in contrast to that of the COPI coat complex, whichcomes as a preassembled heptamer; the GAP activity associated with simultaneousrecruitment of all COPII subunits would preclude stable coat formation. Instead,this two-gear mechanism of GAP stimulation may allow propagation of the coatlattice and membrane curvature to proceed sufficiently to withstand loss of the Sar1

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anchor from the epicenter. Additionally, multivalent interactions of the Sec23-24complex with cargo molecules could further stabilize the outer coat. In support ofthis, gently isolated vesicles generated in the presence of GTP retained substantiallymore Sec23-24 and Sec13-31 than Sar1 (Barlowe et al. 1994).

An additional factor that may promote coat stability is the large peripheral ERprotein, Sec16 (Espenshade et al. 1995). Temperature-sensitive alleles of SEC16block ER to Golgi transport, and are lethal in combination with mutations in SEC12,SEC13, and SEC23 (Kaiser & Schekman 1990). In addition, physical interactionshave been observed between Sec16 and all of the COPII components except Sec13(Espenshade et al. 1995, Gimeno et al. 1996, Supek et al. 2002). In vitro buddingreactions using ER-enriched membranes stripped of Sec16 revealed that vesicleformation was significantly reduced in reactions performed with GTP but not witha nonhydrolyzable GTP analog. Efficient vesicle formation in the GTP reactionswas restored by addition of purified Sec16. The effect of Sec16 on coat dynamicsis not mediated by a suppresion of GTP hydrolysis on Sar1, but may instead reflecta role as a platform to stabilize coat proteins on the membrane despite Sar1-GTPhydrolysis (Supek et al. 2002).

COPI coat recruitment Activated Arf1 is essential to the recruitment of the∼700 kD-heptameric COPI complex from the cytosol (Figure 2; Orci et al.1993), although additional interactions between the COPI subunits and cargo pro-teins such as the p24 family may facilitate membrane association. Under high saltconditions the heptamer dissociates into two functionally distinct subcomplexes,the F-COPI subcomplex (β, γ , ∂ , ζ ) and the B-COPI subcomplex (α, β ′, ε) (Fiedleret al. 1996). The four components of the F-COPI subcomplex are related in se-quence and structure to the clathrin-binding AP complexes (Boehm & Bonifacino2001, Eugster et al. 2000, Hoffman et al. 2003, Watson et al. 2004), and by analogy,the B-COPI subcomplex may function as a structural scaffold in a manner simi-lar to clathrin. In the F-COPI subcomplex, the N-terminal regions of β-COP andγ -COP share sequence homology with the trunk domain of the large AP subunits(e.g., α and β in AP2), and recent structural analysis revealed similarities betweenthe γ -COP C-terminal domain and the α- and β-AP2 appendage domains (Eugsteret al. 2000, Hoffman et al. 2003, Watson et al. 2004). The two other subunits of theF-COPI subcomplex, ∂-COP and ζ -COP, share homology with the µ- and σ -AP2subunits, respectively (Cosson et al. 1996). Although no structural information isavailable for the B-COPI subcomplex, both the α-COP and β ′-COP subunits arepredicted to possess β-propeller WD40 repeats at the N-terminus, which functionin cargo recognition (see below). In yeast, loss of the WD40 domain from eithersubunit is tolerated; however, WD40 deletion from both α-COP and β ′-COP islethal (Eugster et al. 2004).

Interactions between Arf1 and several of the COPI subunits have been doc-umented by photolabile cross-linking (β- and γ -COP) (Zhao et al. 1997, Zhaoet al. 1999) and yeast two-hybrid analysis (β- and ε-COP) (Eugster et al. 2004).Site-specific cross-links between the switch regions of Arf1 and β- and γ -COPare consistent with a requirement for GTP-dependent membrane binding of COPI

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104 LEE ET AL.

(Zhao et al. 1999) and may be structurally analogous to Arf1 binding to the β andγ subunits of AP1 (Austin et al. 2000).

Unlike the COPII coat, where the Sar1 GAP is an integral part of the coat,stimulation of GTP hydrolysis on Arf1 to promote coat dissassembly is not me-diated by a subunit of the coat per se but by a separate ARF GAP. Several dif-ferent ARF GAPs have been identified and all share an essential 70-residue zincfinger domain and a conserved arginine residue, although many participate in non-COPI processes (Donaldson & Jackson 2000). In yeast, the Golgi-localized GAPsGlo3 and Gcs1 appear to have overlapping functions in COPI coat dissassem-bly (Dogic et al. 1999, Poon et al. 1999). The equivalent function in mammaliancells is performed by ARF GAP1, the first ARF GAP to be identified, and pos-sibly other members of the ARF GAP1 family (Nie et al. 2003, Watson et al.2004).

The mechanism by which ARF GAP proteins catalyze GTP hydrolysis onCOPI vesicles remains controversial. Structural analysis of a complex between thecatalytic domain of ARF GAP1 and Arf1-GDP lacking the N-terminal membraneanchor unexpectedly revealed that ARF GAP1 did not supply a catalytic argininefinger at the GTPase active site. Rather, ARF GAP1 appears to stabilize the switch IIregion in a more catalytically favorable configuration (Goldberg 1999). Additionof stoichiometric amounts of COPI further stimulated ARF GAP1 activity bythree orders of magnitude, although it is not known if this is mediated by anarginine finger on COPI (Goldberg 1999). However, under different experimentalconditions, using full-length myristoylated Arf1 in the presence of ARF GAP1and synthetic membranes, addition of COPI did not stimulate GTP hydrolysis(Szafer et al. 2000), although similar experiments with the yeast GAP Glo3p andGolgi membranes yielded a 50-fold stimulation (Szafer et al. 2001). In all cases,the presence of ARF GAP is an essential component to the reaction, as COPIalone has no effect on GTP hydrolysis by Arf1. Furthermore, Arf1, ARF GAP,and COPI are all required for the formation of an aluminum fluoride complex (B.Antonny, personal communication). Aluminum fluoride conventionally acts in a Gprotein–GAP complex to coordinate the arginine finger of the GAP by mimickingthe γ -phosphate of GTP. Thus additional studies that reveal how ARF GAP andthe COPI coat may simultaneously interact with Arf1 should be informative. Inessence, however, the potential effect of COPI on the rate of GTP hydrolysis isbroadly similar to the two-gear mechanism described for the COPII system inthat the greatest GTPase activity is observed only upon assembly of all the coatcomponents.

Given that stable assembly of the COPI coat in the presence of ARF GAP ischallenged by the high rate of GTP hydrolysis on Arf1, how is the coat maintainedto yield productive vesicles? Two potential mechanisms have been discovered forCOPI. Recently, the activity of ARF GAP1 was shown to be sensitive to the degreeof membrane curvature (Bigay et al. 2003). When myrisotylated Arf1 and COPIwere recruited to synthetic liposomes of defined size, Bigay et al. (2003) observedthat the effect of ARF GAP1 on the rate of GTP hydrolysis, and subsequently coat

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dissassembly, increased by two orders of magnitude as the size of the liposomeswas reduced to that of a COPI vesicle. This suggests an elegant mechanism torestrict ARF GAP activity to late in the budding process, preventing prematureuncoating. In particular, ARF GAP would be excluded from regions of negativecurvature such as the vesicle neck. This could potentially result in a vesicle budwith a stable ring of Arf1 and COPI coat at the neck and repeated cycles ofArf1 GTP hydrolysis at the bud tip (Lippincott-Schwartz & Liu 2003). Localizedcycles of binding and release by Arf1 and coat proteins may play a role in cargoconcentration at the bud site (Lanoix et al. 1999).

An additional mechanism to regulate coat stability may be mediated by the cargoproteins themselves. The cytosolic tail of the human hp24a (p24β1) cargo proteinsignificantly inhibited GTP hydrolysis by Arf1, although other p24 members ordi-lysine-retrieval signals had no effect. Slowed GTP hydrolysis may be inducedby hp24a binding to COPI (Goldberg 2000) or ARF GAP1 (Lanoix et al. 2001).The net effect would be to increase uptake of the inhibitory cargo protein andpotentially provide a time window for additional coat-coat interactions and cargocapture to occur.

Cargo Capture

The sorting signals that determine the cellular location of a given protein work byinteracting directly with specific subunits of vesicle coats. By nature, the interac-tion between cargo and coat must be relatively weak because the coat must subse-quently be released from the vesicle to allow membrane fusion with downstreamcompartments. Direct interaction between sorting signals and coat componentshas been demonstrated for many different transport steps, including those betweenthe ER and Golgi. The canonical ER retrieval motif, KKXX, has long been knownas the sorting signal that interacts with the COPI coat to cause ER deposition(Cosson & Letourneur 1994). However, a universal ER export signal has not beendescribed. Instead, a number of different signals seem to govern interaction withthe COPII coat, including di-acidic motifs and hydrophobic/aromatic motifs (Bar-lowe 2003). Our understanding of how these cytoplasmic coats mediate selectivecargo capture has been greatly facilitated by recent structural, biochemical, andgenetic characterization of the interactions between coat and cargo.

CARGO RECOGNITION BY THE COPII SUBUNIT, Sec24 A significant body of evi-dence has implicated the Sec24 subunit of the COPII coat in cargo selection.The Sar1-Sec23-Sec24 complex is the minimal machinery required to interactwith cargo proteins contained in ER membranes (Aridor et al. 1998, Kuehn et al.1998), and inclusion of the Sec24 subunit is required for incorporation of cargo(Miller et al. 2002). The yeast Sec24 homolog, Lst1, is required for ER export ofan abundant plasma membrane protein (Roberg et al. 1999, Shimoni et al. 2000),and vesicles generated with Lst1 in place of Sec24 package a distinct subset ofcargo (Miller et al. 2002). Furthermore, Sec23-24, and in some cases Sec24 alone,

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106 LEE ET AL.

binds in vitro to the cytoplasmic sorting signals of a number of cargo proteins(Mossessova et al. 2003a, Peng et al. 1999, Springer & Schekman 1998).

Molecular details of the interaction between Sec24 and its cargo substrates wererevealed by structural analysis of yeast Sec24 bound to peptides that correspond tospecific sorting signals (Mossessova et al. 2003a). Two sites on Sec24 form inde-pendent cargo-binding pockets that recognize distinct sorting signals (Figure 4).The A-site interacts with a YNNSNPF-containing peptide, one of two binding mo-tifs on the SNARE, Sed5. The B-site recognizes signals found on two different pep-tides: the di-acidic sorting signal of the Golgi protein Sys1 (Votsmeier & Gallwitz2001) and the LXXLE motif of the SNARE, Bet1. Furthermore, the second motifof Sed5, LXXME, competes for interaction with the Bet1 peptide, suggesting italso employs the same site.

The YNNSNPF motif of Sed5 represents a novel class of ER export signal,although its function in Sed5 transport remains to be tested. The peptide binds ina pocket of Sec24 that is membrane-proximal and corresponds spatially to the siteoccupied on Sec23 by Sar1 (Bi et al. 2002, Mossessova et al. 2003a). The capac-ity for additional proteins to bind in this pocket remains to be tested. In contrast,the B-site, located on the opposite side of Sec24, clearly interacts with multiplesequences. Although the same electrostatic interactions are involved in bindingof both the Sys1 and Bet1 motifs, the two peptides show subtly different con-formations (Mossessova et al. 2003a), suggesting that this pocket accommodatessubstrates in multiple binding modes.

The multiplicity of cargo interactions with the B-site was also demonstrated bymutagenesis of this site (Miller et al. 2003). Point mutations in residues that linethis pocket or make electrostatic contact with the Bet1 and Sys1 sorting signals notonly disrupt packaging of Bet1 and Sys1, but also affect additional cargo proteinsto varying degrees, including the p24 family of proteins and the SNAREs, Bos1and Sec22. Of particular interest is the observation that packaging of a secondcargo protein Gap1, which employs a di-acidic sorting signal, is unaffected bythese mutations, suggesting that similar classes of sorting signals may in fact berecognized by distinct sites on the COPII coat. Other experiments to disrupt the B-site on the Sec24 homolog (Lst1) demonstrated that this site is conserved as a cargointeraction domain even though the substrate specificities of the two proteins differ(Miller et al. 2002, 2003). Additional mutagenesis and genetic screening identifieda third cargo-binding site on Sec24 (Miller et al. 2003). Mutation in this C-sitespecifically impairs packaging of the SNARE, Sec22, which is also affected bymutations in the B-site, suggesting a bipartite mode of interaction similar to that ofSed5 (Mossessova et al. 2003a). The molecular details of the interactions betweenSec22 and Sec24 remain to be determined.

The observation that a number of cargo proteins are packaged into vesicles nor-mally in the presence of the mutant forms of Sec24 suggests that additional siteson the COPII coat remain to be identified (Miller et al. 2003). Although it is pos-sible that additional cargo interaction sites will also map to Sec24, a role for eitherSec23 or Sar1 cannot be ruled out in some instances. Several cargo proteins that

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are packaged by both Sec24 and its homolog, Lst1, are unaffected by mutation ofthe B-site in either protein (Miller et al. 2003). These cargoes are good candidatesfor substrates that may interact with Sar1 or Sec23. Indeed, a direct role for Sar1in cargo selection has been implicated by its recruitment to cytoplasmic domainsof cargo proteins in vitro (Belden & Barlowe 2001a, Springer & Schekman 1998)and by its interaction with some cargo proteins within ER membranes (Aridoret al. 2001, Otte & Barlowe 2002). Mammalian glycosyltransferases contain aC-terminal di-arginine motif that is required for ER export; mutation of this motifdisrupts interaction with Sar1, suggesting a major role for Sar1 in selective captureof these proteins (Giraudo & Maccioni 2003). There is less evidence for a role forSec23 in cargo selection, although its structural similarity to Sec24 and moderateconservation of residues important for cargo binding in the Sec24 B-site makeit a prime candidate for further investigation. Together, the multiplicity of cargo-binding sites on Sec24, the presence of Sec24 homologs that expand thecargo repertoire recognized by the COPII coat, and the potential for additionalcargo recognition domains on COPII subunits give an indication of the diversityof cargo substrates that may be accommodated by this coat.

KKXX MOTIFS INTERACT WITH THE COPI COAT Superficially, interaction of theCOPI coat with its cargo substrates seems more straightforward because of theapparent simplicity of the signals involved. However, despite the recognition manyyears ago that KKXX motifs interact directly with the COPI coat, the moleculardetails of this interaction remain obscure (Cosson & Letourneur 1994). Indeed,there is still debate over exactly which subunit of the COPI coat interacts withKKXX motifs. Initial experiments demonstrated that the α/β ′/ε subcomplex bindsto the cytoplasmic tail of a KKXX-containing protein (Cosson & Letourneur 1994).Futhermore, coatomer from sec27-1 (β ′-COP) or ret1-1 (α-COP) mutant yeast cellsfails to bind KKXX signals in vitro (Letourneur et al. 1994). These results areconsistent with further in vitro binding studies that examined coatomer bindingto the cytoplasmic domains of the p24 family of proteins (Fiedler et al. 1996).Binding of the α-, β ′-, and ε-COP subunits is dependent on an intact KKXX motif,whereas the β-, γ -, and ζ -COP subunits bind to a different subset of p24 proteinsand require an aromatic residue. Thus the KKXX-mediated retrieval function wasthought to involve the α/β ′/ε subcomplex while the β/δ/γ /ζ subcomplex woulddirect a separate transport event.

Contradictory evidence came from cross-linking experiments that used a radio-labeled photoreactive KKXX-containing peptide to identify γ -COP as the solebinding partner (Harter et al. 1996, Harter & Wieland 1998). Furthermore, γ -COP also binds to the Rho-related G protein, Cdc42, via a di-lysine motif, aninteraction that may link vesicles to the actin cytoskeleton (Wu et al. 2000). Oneexplanation for these disparate views of cargo binding by the COPI coat is that twoindependent sites recognize the same motif. In support of this, the COPI coat canbe precipitated by neomycin, an antibiotic that contains multivalent pairs of aminogroups (Hudson & Draper 1997). The high degree of cross-linking suggests that

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108 LEE ET AL.

each coatomer complex contains two binding sites for the drug. Drug-dependentprecipitation was inhibited by di-lysine, indicating two di-lysine recognition siteson the COPI coat. Indeed, different di-lysine motifs may interact distinctly with theCOPI coat (Schroder-Kohne et al. 1998), reminiscent of the multiple interactionsof di-acidic motifs with Sec24.

In addition to the two putative KKXX-binding sites, further cargo recognitiondomains remain to be identified on the COPI coat. A yeast two-hybrid screen for in-teracting partners of the δ-COP subunit identified a peptide sequence that can serveas an ER retrieval motif (Cosson et al. 1998). A similar aromatic motif is found onthe ER protein Sec71 and may also correspond to the signal on the p24 proteinsrecognized by the β/δ/γ /ζ -COP subcomplex. Furthermore, the COPI-binding sig-nals on the ER/Golgi SNAREs that cycle between these two compartments alsoremain to be identified. Some yeast COPI mutants show defects in anterogradetransport, thought to be an indirect effect of failure to retrieve anterograde machin-ery (Gaynor & Emr 1997). These subunits are prime candidates for interactingwith the ER-Golgi SNAREs such as Bet1, Bos1, and Sec22.

PRIMING VESICLES FOR FUSION The fundamental importance of the inclusion ofSNAREs in a vesicle to mediate downstream fusion has led to the hypothesis thatthese crucial cargo proteins influence their own uptake by initiating coat polymer-ization (Springer et al. 1999). The observation that the vesicle-associated SNARE,Bet1p, binds in vitro to Sar1p and that this complex subsequently recruits Sec23-24suggests that this essential cargo protein could nucleate coat assembly and therebyensure its incorporation into a nascent vesicle, priming that vesicle for fusion withthe Golgi (Springer & Schekman 1998). However, reconstitution of COPII buddingfrom synthetic liposomes demonstrated that this priming event is not absolutelyrequired to initiate coat assembly (Matsuoka et al. 1998). Furthermore, in vitro ERbudding reactions with the Sec24 homolog, Lst1, generate COPII vesicles that lackBet1, confirming that association with Bet1 is not required to generate a vesicle(Miller et al. 2002). However, the process of cargo selection may still be mecha-nistically coupled to budding such that any abundant cargo protein may nucleatecoat assembly. Such priming of coat recruitment would explain the relatively highlevels of COPII proteins required to generate vesicles from cargo-free syntheticliposomes (Matsuoka et al. 1998). Such a model is supported by the observationthat overexpression of a specific cargo protein rescues defects associated with theCOPI coat and increases the efficiency of vesicle formation (Sandmann et al. 2003).

Although SNAREs are not absolutely required to generate vesicles in vitro,the process of vesicle budding may still be mechanistically coupled to vesiclefusion through incorporation of specific fusion factors in vivo. Further evidencein favor of the vesicle priming hypothesis has come from detailed biochemicalanalysis of the accessibility of the sorting signals of the ER-Golgi SNAREs Bet1and Sed5 (Mossessova et al. 2003a). When Bet1 is assembled in a trans-SNAREbundle, its LXXLE-binding motif is buried, suggesting that monomeric Bet1 is

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preferentially recruited into a COPII vesicle. Conversely, monomeric Sed5 containsan N-terminal regulatory domain that masks the YNNSNPF motif, although theLXXME motif is still available. When Sed5 is assembled into a t-SNARE complexwith Bos1 and Sec22, the N-terminal domain is rearranged such that the YNNSNPFmotif is now accessible, suggesting that this assembly is the preferred COPIIsubstrate. These data suggest a variation of the vesicle priming hypothesis wherebythe COPII coat preferentially selects the fusogenic forms of the SNAREs, therebyprogramming vesicles for fusion either with each other or the Golgi (Mossessovaet al. 2003a).

Priming of vesicles for fusion may not be restricted to SNARE incorporation.Mammalian p115 is one of the tethering factors that helps facilitate vesicle fusionwith the Golgi (see below) and is recruited to COPII vesicles in a manner dependenton the G protein, Rab1. Failure to incorporate p115 results in vesicles that arereduced in their ability to fuse with the Golgi, suggesting that Rab1 primes vesiclesfor fusion by preassembling a tethering factor with its interacting SNARE partners(Allan et al. 2000). Similar priming of COPI vesicles with either SNAREs or tethersto facilitate retrograde transport has not been described, although the observationthat in vitro binding of yeast COPI to Bet1 requires a catalytic activity suppliedby the ARF GAP, Glo3, suggests that a version of priming may also occur in thispathway (Rein et al. 2002).

DELIVERY OF VESICLES: MOTORS, TETHERING,AND FUSION

To complete their journey and fulfill their role as intracellular shuttles, cargo-ladenvesicles must be correctly delivered to and fuse with the appropriate acceptor com-partment. Two separate processes ensure the specificity of this delivery process:vesicle tethering and SNARE assembly. Vesicle tethering anchors the vesicle to thedonor compartment and may prime the SNAREs for subsequent action. Specificcombinations of SNAREs then form a four-helix bundle that drives membranefusion. As described above, the coat proteins may program vesicles for fusion byselecting the appropriate tethers and SNAREs during vesicle budding. Prior to theaction of tethers and SNAREs, vesicles may undergo directed cytoskeleton-drivenmovement to deliver them to the site of fusion.

Motor Proteins and Vesicle Delivery

Although there is little evidence for a role for the cytoskeleton in yeast ER-Golgitransport, this step in mammalian cells is sensitive to microtubule disruption(Malaisse & Orci 1979, Presley et al. 1997). In mammalian cells, COPII vesi-cles that bud from the ER may be first delivered to the ERGIC/VTC, most likelythrough a combination of homotypic and heterotypic fusion. This intermediatecompartment then relocates to the perinuclear Golgi ribbon in a manner that is

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110 LEE ET AL.

dependent on the microtubule cytoskeleton and the motor protein dynein/dynactin(Presley et al. 1997, Scales et al. 1997). Thus the microtubule dependence ofER-Golgi transport may lie both in the movement of COPII vesicles away fromthe ER, and in the delivery of the ERGIC to the Golgi proper. Perhaps the presenceof an intermediate compartment between the ER and Golgi in mammalian cells isthe result of the long-range transport that must occur between dispersed ER exitsites and the central peri-nuclear Golgi. Yeast cells, being much smaller, may relyinstead on simple diffusion to generate encounters between ER-derived vesiclesand single dispersed Golgi elements (Preuss et al. 1992). By contrast, plant cells,which contain a mobile pool of Golgi stacks, may employ the coordinated move-ment of both organelles such that ER exit sites are juxtaposed with an individualGolgi stack, precluding the requirement for microtubules in ER-Golgi transport(Brandizzi et al. 2002).

The actin cytoskeleton seems likely to play a role in retrograde traffic of COPIvesicles to the ER. Numerous actin-binding proteins and actin itself are located onthe Golgi and are further recruited in response to activation of ARF (Fucini et al.2000, Godi et al. 1998). Additional interactions between the COPI coat and the actincytoskeleton are likely to involve the Rho-related G protein, Cdc42, which bindsto γ -COPI (Wu et al. 2000) and is thought to modulate actin dynamics through theArp2/3 complex to effect Golgi-ER transport (Luna et al. 2002). However, whetherthis coat-dependent actin assembly is involved in transport of vesicles to the ER,or instead serves to regulate vesicle fission, remains to be determined (Fucini et al.2002, Stamnes 2002).

Vesicle Tethering

The first interaction that a vesicle has with its acceptor compartment is likely tobe mediated by a large protein assembly that tethers the vesicle to the membrane.The mechanistic details of how tethers contribute to vesicle fusion remain obscure;they may merely increase the probability of SNARE encounter or may functioncatalytically to rearrange SNARE complexes to facilitate fusion. Indeed, differentsets of tethering complexes may perform each of these functions (Whyte & Munro2002). In the anterograde pathway, diffusible vesicles become anchored to Golgimembranes by the addition of Uso1, the yeast homolog of p115 (Barlowe 1997,Cao et al. 1998). Uso1/p115 is a large coiled-coil protein that forms an extendedoligomeric structure (Sapperstein et al. 1995), possibly facilitating SNARE com-plex assembly (Sapperstein et al. 1996, Shorter et al. 2002). In addition to Uso1,a second large oligomeric complex is also likely to function in vesicle tethering.The TRAPP I (transport protein particle I) complex contains seven subunits and isthought to be the landmark on the Golgi that attracts COPII vesicles (Sacher et al.1998, 2001). TRAPP also functions in vitro as a GEF for the yeast Rab1 homolog,Ypt1 (Jones et al. 2000, Wang et al. 2000), which is responsible for the recruitmentof p115/Uso1 to membranes (Allan et al. 2000, Cao et al. 1998). Thus the tethering

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of COPII vesicles to Golgi membranes is likely to involve initial binding of COPIIvesicles via TRAPP, which would activate Ypt1 to recruit Uso1. Binding of Uso1would stabilize the association of the vesicle with the membrane and also facilitateSNARE assembly, thereby leading to membrane fusion and delivery of the vesiclecontents. An additional putative tethering assembly located on the Golgi, the con-served oligomeric Golgi (COG) complex, has been implicated in early transportsteps in the secretory pathway. However, the vesicles that dock to this complexare unknown, and the ER-Golgi transport defects associated with mutations in thiscomplex are likely to be indirect effects of disrupting Golgi function (Whyte &Munro 2002).

The tethering factors that link COPI vesicles to the ER membrane remain poorlycharacterized. The best candidate is the yeast Dsl1, a peripheral membrane proteinthat localizes to the ER. Dsl1 exists in an oligomeric complex that also containsTip20, which in turn interacts with Sec20 and Ufe1, two of the retrograde SNAREs,(Lewis et al. 1997, Reilly et al. 2001). Dsl1 is an essential protein and certain dsl1mutants show specific retrograde transport defects (Reilly et al. 2001). Furthercharacterization of the Dsl1-containing complex and its interaction with the Golgi-ER SNAREs should shed further light on how this putative tether may facilitatemembrane fusion.

SNARE Assembly and Fusion

The final step in vesicle-mediated transport between organelles is fusion of thevesicle with the acceptor compartment. This process is mediated by transmem-brane SNARE proteins that interact in specific combinations to bring the vesicleand acceptor membranes into close proximity and drive fusion. The cytoplasmicdomains of SNAREs assemble into a four-helix bundle, generally with each he-lix contributed by a single transmembrane protein. Thus a combination of fourdifferent SNAREs is required to drive membrane fusion. In vitro fusion experi-ments with SNAREs reconstituted into synthetic liposomes have been instrumentalin defining the specific combinations of SNAREs that mediate membrane fusion(Fukuda et al. 2000, McNew et al. 2000, Parlati et al. 2000). This reconstitutionhas yielded a model for SNARE assembly whereby the target membrane SNARE(t-SNARE) has three subunits: a syntaxin-like heavy chain and two light chainscomposed of either one or two additional SNAREs, depending on the number ofcoiled-coil domains. The vesicle-SNARE (v-SNARE) is a monomeric protein thatmust be on the membrane opposite from the t-SNARE assembly (Fukuda et al.2000). Testing all possible combinations of the yeast SNAREs, only the specificcombination of the t-SNARE Sed5/Bos1/Sec22 and the v-SNARE Bet1 resultedin liposome fusion (Parlati et al. 2000). This is somewhat contradictory to in vitrobudding and fusion experiments that reconstitute transport between the ER andGolgi. In these experiments, Sed5 was required on the Golgi acceptor membranes,whereas both Bet1 and Bos1 were required on the ER-derived vesicle (Cao &

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Barlowe 2000, Spang & Schekman 1998). In the retrograde Golgi-ER pathway,both Sec22 and Bet1 were implicated as the v-SNAREs (Spang & Schekman 1998).It is possible that the mutant membranes used in these in vitro budding and fusionassays are subject to pleiotropic effects, making the precise role of each SNAREdifficult to parse out. The SNAREs of the retrograde pathway have been character-ized by genetic and biochemical analysis. Ufe1 is the syntaxin-like SNARE of theER membrane, required for both homotypic ER fusion and retrograde transport(Lewis et al. 1997, Patel et al. 1998). The other SNARE components that interactwith Ufe1 include Sec22, Sec20, and Use1/Slt1 (Burri et al. 2003, Dilcher et al.2003, Lewis et al. 1997), although the topology of these SNAREs remains to betested using the liposome fusion assay.

In addition to the SNAREs themselves, a class of regulatory proteins plays animportant but ill-defined role in membrane fusion. Proteins of the Sec1/Munc18family (SM) interact with syntaxin-like SNARE proteins to modulate SNAREactivity. It is still controversial whether SM proteins act as positive or nega-tive regulators of membrane fusion, and it is possible that different mechanismsfunction at various steps of membrane transport in different systems (Toonen &Verhage 2003). The yeast protein, Sly1, is an SM protein that binds to both Ufe1and Sed5 via a short motif upstream of the N-terminal regulatory domain of thesesyntaxin-like SNAREs (Yamaguchi et al. 2002). Sly1 seems to promote the as-sembly of Sed5 into the correct v-t-SNARE complex (Peng & Gallwitz 2002).Structural analysis of the binding of Sly1 to the Sed5 motif suggests that the inter-action between these two proteins occupies a very small binding surface on Sed5,leaving substantial room for additional protein-protein interactions that could con-tribute to SNARE assembly, membrane fusion, and disassembly of SNAREs afterfusion (Bracher & Weissenhorn 2002). Thus membrane fusion is mediated by nu-merous proteins including the Rabs, which act with tethering complexes to dockvesicles to the target membrane, SNAREs, which interact in specific combina-tions to drive membrane fusion, and the SM proteins, which also contribute tospecificity.

NON-CANONICAL TRANSPORT

Given the diversity of traffic through the secretory pathway, it is perhaps not sur-prising that additional mechanisms exist to deal with particular cargo molecules.Although the majority of secreted proteins are small enough to be comfortablyenclosed in a 60–80-nm COPII vesicle, several cell types secrete especially largeprotein and lipid macromolecular complexes. In particular, the transport of lipopar-ticles and procollagen fibers would challenge the dimensions of a typical vesicle.In addition, some lipid species such as ceramide can reach the Golgi through anonvesicular route (Funato & Riezman 2001, Hanada et al. 2003). Finally, a subsetof Golgi resident proteins, as well as some bacterial toxins, may reach the ER viaa COPI-independent mechanism.

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Anterograde Transport of Large Cargo

Secretion of both apoB-containing very low density lipoproteins (VLDL) fromhepatic cells and chylomicrons from intestinal cells is of considerable interestbecause of their relevance to cardiovascular disease. Hereditary mutations in anisoform of Sar1 (SARA2) are associated with intracellular accumulation of chy-lomicron particles (Jones et al. 2003), suggesting a role for COPII despite thelarge size of chylomicrons (150–500 nm; Siddiqi et al. 2003) and VLDL particles(80–200 nm; Gusarova et al. 2003). In addition, expression of a dominant-negativeform of Sar1 (Sar1T39A) in VLDL-secreting rat hepatoma cells resulted in apoBretention in the ER. In vitro reconstitution of apoB100-VLDL export from theER of hepatic cells suggests that full lipidation to the mature VLDL particle mayoccur in post-ER compartments, at least partially relieving the size burden on ERexport (Gusarova et al. 2003). However, a separate study that used a cell-free as-say to examine the formation of prechylomicron transport vesicles from the ER ofintestinal epithelial cells observed the release of large (350–500 nm) membrane-enclosed particles (Siddiqi et al. 2003). Although COPII proteins were presenton the purified prechylomicron vesicles, depletion of Sar1 or Sec31 unexpectedlystimulated prechylomicron release but prevented fusion with the Golgi (Siddiqiet al. 2003). Thus the precise role of COPII in the generation of large lipoparticlecarriers remains unclear.

Procollagen assembles in the ER into fibers >300 nm in length, and procol-lagen secretion has been used as a tool to examine the role of small vesicles inanterograde intra-Golgi and ER-Golgi transport (Bonfanti et al. 1998, Mironovet al. 2003, Stephens & Pepperkok 2002). As with lipoprotein export, microin-jection of a dominant-negative form of Sar1 blocked exit of procollagen fromthe ER (Mironov et al. 2003, Stephens & Pepperkok 2002). However, it is notknown whether a potentially flexible COPII coat directly contributes to the for-mation of specialized larger transport carriers (Stephens & Pepperkok 2002) oracts only indirectly at sites adjacent to procollagen protrusion (Mironov et al.2003). Development of a cell-free export assay for procollagen export maydelineate the exact contribution of cytosolic coat proteins to procollagen carrierformation.

COPI-Independent Retrograde Transport

Retrieval of proteins with di-lysine and KDEL-sorting signals is clearly dependenton COPI function. However, the existence of a COPI-independent retrieval path-way is suggested by the fact that certain protein toxins, which lack any obviousretrieval motifs, can be delivered to the ER (Storrie et al. 2000). Inhibition of COPIfunction by microinjection of anti-COPI antibodies or expression of a dominant-negative form of Arf1 fails to block the delivery of Shiga toxin and resident Golgiglycosyltransferases to the ER. Under the same conditions, the retrieval of thedi-lysine-containing cargo protein, ERGIC53, and the KDEL receptor is inhibitedas expected (Girod et al. 1999). Furthermore, the converse effect is observed upon

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114 LEE ET AL.

expression of a GDP-restricted Rab6, which inhibits retrieval of Shiga toxin andglycosyltransferases but not ERGIC53 and KDEL receptor (Girod et al. 1999).Rab6 may act to recruit the dynein-dynactin motor proteins through interactionwith BICD1 and 2, the mammalian orthologs of Drosophila Bicaudal-D (Mataniset al. 2002). In comparison with the kinetics of COPI transport, COPI-independentrecycling occurs slowly (Girod et al. 1999), and the nature of the transport vehicleis not known. This alternate pathway may function to return long-lived Golgi gly-cosylation enzymes back to the ER for quality surveillance or may be importantfor lipid recycling (Storrie et al. 2000).

CONCLUSIONS

One of the ongoing challenges in cell biology is to understand the moleculardetails that allow cytosolic coat proteins to fulfill their dual roles in membranedeformation and cargo capture. Structural details of the coat machinery have al-ready provided many insights and will continue to be particularly informative. Thesimilarities between the COPI coat and the clathrin AP complexes, which mediatetrafficking in the late secretory pathway, are becoming increasingly clear, and itwill be interesting to see if the resemblance extends to cargo- and effector-bindingsites as these are uncovered. For both COPI and COPII, the contribution of theouter coat, i.e., the α/β ′/ε subcomplex for COPI and Sec13-Sec31 for COPII, isstill somewhat mysterious, and awaits biochemical and structural clues about theirrole in propagating the coat lattice on the membrane.

The efficient capture of most cargo proteins requires interactions with the vesiclecoat, either directly or through adaptors. Identification of new protein export andretrieval signals and adaptors, in combination with an analysis of cargo-bindingsite mutants on the coat, will help to classify these sorting signals. It will also beimportant to understand the exceptions to the rule. What role does the COPII ma-chinery play in the anterograde transport of large macromolecular complexes, andwhat is the machinery required to direct cargo proteins into the COPI-independentretrograde route? Identification of specific mutants that disrupt these processes incombination with cell-free assays and microscopic imaging should be informative.

Finally, much is yet to be discovered on how transport is regulated: in particu-lar, how the transition between protein folding and export is sensed; how sites ofcoat recruitment and vesicle budding are marked; how progress during membranedeformation is monitored; and how vesicle uncoating prior to fusion progresses.Although many new molecules that perform these functions no doubt await dis-covery, many others may already be familiar players with as yet unappreciatedroles.

ACKNOWLEDGMENTS

We are grateful to Pierre Cosson for critical comments on the manuscript and toBruno Antonny for helpful discussions and communicating unpublished results.

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Work in the authors’ laboratories is sponsored by the Howard Hughes MedicalInstitute (R.S. and J.G.) and by the Swiss National Science Foundation (L.O.).

The Annual Review of Cell and Developmental Biology is online athttp://cellbio.annualreviews.org

LITERATURE CITED

Allan BB, Moyer BD, Balch WE. 2000. Rab1recruitment of p115 into a cis-SNARE com-plex: programming budding COPII vesiclesfor fusion. Science 289:444–48

Amor JC, Harrison DH, Kahn RA, RingeD. 1994. Structure of the human ADP-ribosylation factor 1 complexed with GDP.Nature 372:704–8

Antonny B, Huber I, Paris S, Chabre M,Cassel D. 1997. Activation of ADP-ribo-sylation factor 1 GTPase-activating proteinby phosphatidylcholine-derived diacylglyc-erols. J. Biol. Chem. 272:30848–51

Antonny B, Madden D, Hamamoto S, Orci L,Schekman R. 2001. Dynamics of the COPIIcoat with GTP and stable analogues. Nat.Cell Biol. 3:531–37

Appenzeller C, Andersson H, Kappeler F, HauriHP. 1999. The lectin ERGIC-53 is a cargotransport receptor for glycoproteins. Nat.Cell Biol. 1:330–34

Aridor M, Fish KN, Bannykh S, Weissman J,Roberts TH, et al. 2001. The Sar1 GTPasecoordinates biosynthetic cargo selection withendoplasmic reticulum export site assembly.J. Cell Biol. 152:213–29

Aridor M, Weissman J, Bannykh S, Nuof-fer C, Balch WE. 1998. Cargo selection bythe COPII budding machinery during exportfrom the ER. J. Cell Biol. 141:61–70

Austin C, Hinners I, Tooze SA. 2000. Di-rect and GTP-dependent interaction ofADP-ribosylation factor 1 with clathrinadaptor protein AP-1 on immature secre-tory granules. J. Biol. Chem. 275:21862–69

Baker EK, Colley NJ, Zuker CS. 1994. Thecyclophilin homolog NinaA functions as achaperone, forming a stable complex in vivo

with its protein target rhodopsin. EMBO J.13:4886–95

Barlowe C. 1997. Coupled ER to Golgi trans-port reconstituted with purified cytosolic pro-teins. J. Cell Biol. 139:1097–108

Barlowe C. 2003. Signals for COPII-dependentexport from the ER: what’s the ticket out?Trends Cell Biol. 13:295–300

Barlowe C, Orci L, Yeung T, Hosobuchi M,Hamamoto S, et al. 1994. COPII: a mem-brane coat formed by Sec proteins that drivevesicle budding from the endoplasmic retic-ulum. Cell 77:895–907

Barlowe C, Schekman R. 1993. SEC12 encodesa guanine-nucleotide-exchange factor essen-tial for transport vesicle budding from theER. Nature 365:347–49

Belden WJ, Barlowe C. 2001a. Distinct rolesfor the cytoplasmic tail sequences of Emp24pand Erv25p in transport between the en-doplasmic reticulum and Golgi complex. J.Biol. Chem. 276:43040–48

Belden WJ, Barlowe C. 2001b. Role ofErv29p in collecting soluble secretory pro-teins into ER-derived transport vesicles. Sci-ence 294:1528–31

Bi X, Corpina RA, Goldberg J. 2002. Structureof the Sec23/24-Sar1 pre-budding complexof the COPII vesicle coat. Nature 419:271–77

Bigay J, Gounon P, Robineau S, Antonny B.2003. Lipid packing sensed by ArfGAP1couples COPI coat disassembly to membranebilayer curvature. Nature 426:563–66

Boehm M, Bonifacino JS. 2001. Adaptins: thefinal recount. Mol. Biol. Cell 12:2907–20

Bonfanti L, Mironov AA Jr, Martinez-Menarguez JA, Martella O, Fusella A, et al.1998. Procollagen traverses the Golgi stack

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.


116 LEE ET AL.

without leaving the lumen of cisternae: evi-dence for cisternal maturation. Cell 95:993–1003

Bracher A, Weissenhorn W. 2002. Structuralbasis for the Golgi membrane recruitment ofSly1p by Sed5p. EMBO J. 21:6114–24

Brandizzi F, Snapp EL, Roberts AG, Lippin-cott-Schwartz J, Hawes C. 2002. Membraneprotein transport between the endoplasmicreticulum and the Golgi in tobacco leaves isenergy dependent but cytoskeleton indepen-dent: evidence from selective photobleach-ing. Plant Cell 14:1293–309

Burri L, Varlamov O, Doege CA, HofmannK, Beilharz T, et al. 2003. A SNARE re-quired for retrograde transport to the en-doplasmic reticulum. Proc. Natl. Acad. Sci.USA 100:9873–77

Cabrera M, Muniz M, Hidalgo J, Vega L, MartinME, Velasco A. 2003. The retrieval functionof the KDEL receptor requires PKA phos-phorylation of its C-terminus. Mol. Biol. Cell14:4114–25

Caldwell SR, Hill KJ, Cooper AA. 2001. Degra-dation of endoplasmic reticulum (ER) qualitycontrol substrates requires transport betweenthe ER and Golgi. J. Biol. Chem. 276:23296–303

Cao X, Ballew N, Barlowe C. 1998. Ini-tial docking of ER-derived vesicles requiresUso1p and Ypt1p but is independent ofSNARE proteins. EMBO J. 17:2156–65

Cao X, Barlowe C. 2000. Asymmetric require-ments for a Rab GTPase and SNARE pro-teins in fusion of COPII vesicles with ac-ceptor membranes. J. Cell Biol. 149:55–66

Chardin P, Callebaut I. 2002. The yeast Sar ex-change factor Sec12, and its higher organismorthologs, fold as beta-propellers. FEBS Lett.525:171–73

Colley NJ, Baker EK, Stamnes MA, Zuker CS.1991. The cyclophilin homolog ninaA is re-quired in the secretory pathway. Cell 67:255–63

Cosson P, Demolliere C, Hennecke S, Du-den R, Letourneur F. 1996. Delta- and zeta-COP, two coatomer subunits homologous to

clathrin-associated proteins, are involved inER retrieval. EMBO J. 15:1792–98

Cosson P, Lefkir Y, Demolliere C, LetourneurF. 1998. New COPI-binding motifs involvedin ER retrieval. EMBO J. 17:6863–70

Cosson P, Letourneur F. 1994. Coatomer inter-action with di-lysine endoplasmic reticulumretention motifs. Science 263:1629–31

Dilcher M, Veith B, Chidambaram S, HartmannE, Schmitt HD, Fischer von Mollard G. 2003.Use1p is a yeast SNARE protein requiredfor retrograde traffic to the ER. EMBO J.22:3664–74

Dogic D, de Chassey B, Pick E, Cassel D, LefkirY, et al. 1999. The ADP-ribosylation factorGTPase-activating protein Glo3p is involvedin ER retrieval. Eur. J. Cell Biol. 78:305–10

Donaldson JG, Jackson CL. 2000. Regulatorsand effectors of the ARF GTPases. Curr.Opin. Cell Biol. 12:475–82

Dwyer ND, Troemel ER, Sengupta P, Barg-mann CI. 1998. Odorant receptor localiza-tion to olfactory cilia is mediated by ODR-4,a novel membrane-associated protein. Cell93:455–66

Elrod-Erickson MJ, Kaiser CA. 1996. Genesthat control the fidelity of endoplasmic retic-ulum to Golgi transport identified as suppres-sors of vesicle budding mutations. Mol. Biol.Cell 7:1043–58

Espenshade P, Gimeno RE, Holzmacher E, Te-ung P, Kaiser CA. 1995. Yeast SEC16 geneencodes a multidomain vesicle coat pro-tein that interacts with Sec23p. J. Cell Biol.131:311–24

Eugster A, Frigerio G, Dale M, DudenR. 2000. COP I domains required forcoatomer integrity, and novel interactionswith ARF and ARF-GAP. EMBO J. 19:3905–17

Eugster A, Frigerio G, Dale M, Duden R. 2004.The α- and β ′-COP WD40 domains medi-ate cargo-selective interactions with distinctdi-lysine motifs. Mol. Biol. Cell 15:1011–23

Fiedler K, Veit M, Stamnes MA, Rothman JE.1996. Bimodal interaction of coatomer withthe p24 family of putative cargo receptors.Science 273:1396–99

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.



Fucini RV, Chen JL, Sharma C, Kessels MM,Stamnes M. 2002. Golgi vesicle proteins arelinked to the assembly of an actin complexdefined by mAbp1. Mol. Biol. Cell 13:621–31

Fucini RV, Navarrete A, Vadakkan C, LacomisL, Erdjument-Bromage H, et al. 2000. Ac-tivated ADP-ribosylation factor assemblesdistinct pools of actin on Golgi membranes.J. Biol. Chem. 275:18824–29

Fukuda R, McNew JA, Weber T, Parlati F, En-gel T, et al. 2000. Functional architecture ofan intracellular membrane t-SNARE. Nature407:198–202

Funato K, Riezman H. 2001. Vesicular and non-vesicular transport of ceramide from ER tothe Golgi apparatus in yeast. J. Cell Biol.155:949–59

Garcia-Mata R, Szul T, Alvarez C, SztulE. 2003. ADP-ribosylation factor/COPI-dependent events at the endoplasmic reti-culum-Golgi interface are regulated by theguanine nucleotide exchange factor GBF1.Mol. Biol. Cell 14:2250–61

Gaynor EC, Emr SD. 1997. COPI-independentanterograde transport: cargo-selective ER toGolgi protein transport in yeast COPI mu-tants. J. Cell Biol. 136:789–802

Gilstring CF, Melin-Larsson M, LjungdahlPO. 1999. Shr3p mediates specific COPIIcoatomer-cargo interactions required for thepackaging of amino acid permeases into ER-derived transport vesicles. Mol. Biol. Cell.10:3549–65

Gimeno RE, Espenshade P, Kaiser CA. 1996.COPII coat subunit interactions: Sec24p andSec23p bind to adjacent regions of Sec16p.Mol. Biol. Cell 7:1815–23

Giraudo CG, Maccioni HJ. 2003. Endoplasmicreticulum export of glycosyltransferases de-pends on interaction of a cytoplasmic dibasicmotif with Sar1. Mol. Biol. Cell 14:3753–66

Girod A, Storrie B, Simpson JC, Johannes L,Goud B, et al. 1999. Evidence for a COP-I-independent transport route from the Golgicomplex to the endoplasmic reticulum. Nat.Cell Biol. 1:423–30

Godi A, Santone I, Pertile P, Devarajan P,

Stabach PR, et al. 1998. ADP ribosyla-tion factor regulates spectrin binding to theGolgi complex. Proc. Natl. Acad. Sci. USA95:8607–12

Goldberg J. 1998. Structural basis for ac-tivation of ARF GTPase: mechanisms ofguanine nucleotide exchange and GTP-myristoyl switching. Cell 95:237–48

Goldberg J. 1999. Structural and functionalanalysis of the ARF1-ARFGAP complex re-veals a role for coatomer in GTP hydrolysis.Cell 96:893–902

Goldberg J. 2000. Decoding of sorting signalsby coatomer through a GTPase switch in theCOPI coat complex. Cell 100:671–79

Gommel DU, Memon AR, Heiss A, LottspeichF, Pfannstiel J, et al. 2001. Recruitment toGolgi membranes of ADP-ribosylation fac-tor 1 is mediated by the cytoplasmic domainof p23. EMBO J. 20:6751–60

Gusarova V, Brodsky JL, Fisher EA. 2003.Apolipoprotein B100 exit from the endoplas-mic reticulum (ER) is COPII-dependent, andits lipidation to very low density lipoproteinoccurs post-ER. J. Biol. Chem. 278:48051–58

Hammond C, Helenius A. 1994. Folding ofVSV G protein: sequential interaction withBiP and calnexin. Science 266:456–58

Hanada K, Kumagai K, Yasuda S, Miura Y,Kawano M, et al. 2003. Molecular machin-ery for non-vesicular trafficking of ceramide.Nature 426:803–9

Harter C, Pavel J, Coccia F, Draken E, Wege-hingel S, et al. 1996. Nonclathrin coat pro-tein gamma, a subunit of coatomer, binds tothe cytoplasmic dilysine motif of membraneproteins of the early secretory pathway. Proc.Natl. Acad. Sci. USA 93:1902–6

Harter C, Wieland FT. 1998. A single bind-ing site for dilysine retrieval motifs and p23within the gamma subunit of coatomer. Proc.Natl. Acad. Sci. USA 95:11649–54

Hoffman GR, Rahl PB, Collins RN, CerioneRA. 2003. Conserved structural motifs inintracellular trafficking pathways: structureof the gammaCOP appendage domain. Mol.Cell 12:615–25

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.


118 LEE ET AL.

Huang M, Weissman JT, Beraud-Dufour S,Luan P, Wang C, et al. 2001. Crystal struc-ture of Sar1-GDP at 1.7 A resolution and therole of the NH2 terminus in ER export. J. CellBiol. 155:937–48

Hudson RT, Draper RK. 1997. Interaction ofcoatomer with aminoglycoside antibiotics:evidence that coatomer has at least two dily-sine binding sites. Mol. Biol. Cell 8:1901–10

Jackson CL, Casanova JE. 2000. Turning onARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell. Biol. 10:60–67

Jones B, Jones EL, Bonney SA, Patel HN,Mensenkamp AR, et al. 2003. Mutations ina Sar1 GTPase of COPII vesicles are asso-ciated with lipid absorption disorders. Nat.Genet. 34:29–31

Jones S, Newman C, Liu F, Segev N. 2000.The TRAPP complex is a nucleotide ex-changer for Ypt1 and Ypt31/32. Mol. Biol.Cell 11:4403–11

Kaiser C. 2000. Thinking about p24 proteinsand how transport vesicles select their cargo.Proc. Natl. Acad. Sci. USA 97:3783–85

Kaiser CA, Schekman R. 1990. Distinct sets ofSEC genes govern transport vesicle forma-tion and fusion early in the secretory path-way. Cell 61:723–33

Kim PS, Arvan P. 1998. Endocrinopathies in thefamily of endoplasmic reticulum (ER) stor-age diseases: disorders of protein traffickingand the role of ER molecular chaperones. En-docr. Rev. 19:173–202

Kuehn MJ, Herrmann JM, Schekman R. 1998.COPII-cargo interactions direct protein sort-ing into ER-derived transport vesicles. Na-ture 391:187–90

Kuehn MJ, Schekman R, Ljungdahl PO. 1996.Amino acid permeases require COPII com-ponents and the ER resident membraneprotein Shr3p for packaging into transportvesicles in vitro. J. Cell Biol. 135:585–95

Kuge O, Dascher C, Orci L, Rowe T, AmherdtM, et al. 1994. Sar1 promotes vesicle bud-ding from the endoplasmic reticulum but not

Golgi compartments. J. Cell Biol. 125:51–65

Lanoix J, Ouwendijk J, Lin CC, Stark A, LoveHD, et al. 1999. GTP hydrolysis by ARF-1mediates sorting and concentration of Golgiresident enzymes into functional COP I vesi-cles. EMBO J. 18:4935–48

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cas-sel D, et al. 2001. Sorting of Golgi resi-dent proteins into different subpopulations ofCOPI vesicles: a role for ArfGAP1. J. CellBiol. 155:1199–212

Lau WT, Howson RW, Malkus P, SchekmanR, O’Shea EK. 2000. Pho86p, an endoplas-mic reticulum (ER) resident protein in Sac-charomyces cerevisiae, is required for ERexit of the high-affinity phosphate transporterPho84p. Proc. Natl. Acad. Sci. USA 97:1107–12

Lederkremer GZ, Cheng Y, Petre BM, Vo-gan E, Springer S, et al. 2001. Structureof the Sec23p/24p and Sec13p/31p com-plexes of COPII. Proc. Natl. Acad. Sci. USA98:10704–9

Letourneur F, Gaynor EC, Hennecke S, De-molliere C, Duden R, et al. 1994. Coatomeris essential for retrieval of dilysine-taggedproteins to the endoplasmic reticulum. Cell79:1199–207

Letourneur F, Hennecke S, Demolliere C, Cos-son P. 1995. Steric masking of a dilysine en-doplasmic reticulum retention motif duringassembly of the human high affinity receptorfor immunoglobulin E. J. Cell Biol. 129:971–78

Lewis MJ, Pelham HR. 1992. Ligand-inducedredistribution of a human KDEL receptorfrom the Golgi complex to the endoplasmicreticulum. Cell 68:353–64

Lewis MJ, Rayner JC, Pelham HR. 1997. Anovel SNARE complex implicated in vesi-cle fusion with the endoplasmic reticulum.EMBO J. 16:3017–24

Lippincott-Schwartz J, Liu W. 2003. Membranetrafficking: coat control by curvature. Nature426:507–8

Luna A, Matas OB, Martinez-Menarguez JA,Mato E, Duran JM, et al. 2002. Regulation

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.



of protein transport from the Golgi com-plex to the endoplasmic reticulum by CDC42and N-WASP. Mol. Biol. Cell 13:866–79

Ma D, Zerangue N, Raab-Graham K, Fried SR,Jan YN, Jan LY. 2002. Diverse traffickingpatterns due to multiple traffic motifs in Gprotein-activated inwardly rectifying potas-sium channels from brain and heart. Neuron33:715–29

Majoul I, Straub M, Hell SW, Duden R, Sol-ing HD. 2001. KDEL-cargo regulates inter-actions between proteins involved in COPIvesicle traffic: measurements in living cellsusing FRET. Dev. Cell 1:139–53

Malaisse WJ, Orci L. 1979. The role of thecytoskeleton in pancreatic B-cell function.Meth. Achiev. Exp. Pathol. 9:112–36

Malkus P, Jiang F, Schekman R. 2002. Con-centrative sorting of secretory cargo pro-teins into COPII-coated vesicles. J. Cell Biol.159:915–21

Mallabiabarrena A, Fresno M, Alarcon B. 1992.An endoplasmic reticulum retention signal inthe CD3 epsilon chain of the T-cell receptor.Nature 357:593–96

Margeta-Mitrovic M, Jan YN, Jan LY. 2000.A trafficking checkpoint controls GABA(B)receptor heterodimerization. Neuron 27:97–106

Martinez-Menarguez JA, Geuze HJ, Slot JW,Klumperman J. 1999. Vesicular tubular clus-ters between the ER and Golgi mediate con-centration of soluble secretory proteins byexclusion from COPI-coated vesicles. Cell98:81–90

Matanis T, Akhmanova A, Wulf P, Del Nery E,Weide T, et al. 2002. Bicaudal-D regulatesCOPI-independent Golgi-ER transport by re-cruiting the dynein-dynactin motor complex.Nat. Cell Biol. 4:986–92

Matsuoka K, Orci L, Amherdt M, Bednarek SY,Hamamoto S, et al. 1998. COPII-coated vesi-cle formation reconstituted with purified coatproteins and chemically defined liposomes.Cell 93:263–75

Matsuoka K, Schekman R, Orci L, HeuserJE. 2001. Surface structure of the COPII-

coated vesicle. Proc. Natl. Acad. Sci. USA98:13705–9

McNew JA, Parlati F, Fukuda R, Johnston RJ,Paz K, et al. 2000. Compartmental speci-ficity of cellular membrane fusion encodedin SNARE proteins. Nature 407:153–59

Mezzacasa A, Helenius A. 2002. The transi-tional ER defines a boundary for quality con-trol in the secretion of tsO45 VSV glycopro-tein. Traffic 3:833–49

Miller E, Antonny B, Hamamoto S, SchekmanR. 2002. Cargo selection into COPII vesiclesis driven by the Sec24p subunit. EMBO J.21:6105–13

Miller EA, Beilharz TH, Malkus PN, Lee MC,Hamamoto S, et al. 2003. Multiple cargobinding sites on the COPII subunit Sec24pensure capture of diverse membrane proteinsinto transport vesicles. Cell 114:497–509

Mironov AA, Mironov AA Jr, BeznoussenkoGV, Trucco A, Lupetti P, et al. 2003. ER-to-Golgi carriers arise through direct en blocprotrusion and multistage maturation of spe-cialized ER exit domains. Dev. Cell 5:583–94

Mossessova E, Bickford LC, Goldberg J. 2003a.SNARE selectivity of the COPII coat. Cell114:483–95

Mossessova E, Corpina RA, Goldberg J. 2003b.Crystal structure of ARF1∗Sec7 complexedwith Brefeldin A and its implications for theguanine nucleotide exchange mechanism.Mol. Cell 12:1403–11

Mossessova E, Gulbis JM, Goldberg J. 1998.Structure of the guanine nucleotide exchangefactor Sec7 domain of human arno and analy-sis of the interaction with ARF GTPase. Cell92:415–23

Muniz M, Morsomme P, Riezman H. 2001. Pro-tein sorting upon exit from the endoplasmicreticulum. Cell 104:313–20

Muniz M, Nuoffer C, Hauri HP, Riezman H.2000. The Emp24 complex recruits a specificcargo molecule into endoplasmic reticulum-derived vesicles. J. Cell Biol. 148:925–30

Nehls S, Snapp EL, Cole NB, Zaal KJ, Ken-worthy AK, et al. 2000. Dynamics and re-tention of misfolded proteins in native ERmembranes. Nat. Cell Biol. 2:288–95

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.


120 LEE ET AL.

Nie Z, Hirsch DS, Randazzo PA. 2003. Arf andits many interactors. Curr. Opin. Cell Biol.15:396–404

Nufer O, Kappeler F, Guldbrandsen S, HauriHP. 2003. ER export of ERGIC-53 is con-trolled by cooperation of targeting determi-nants in all three of its domains. J. Cell Sci.116:4429–40

O’Kelly I, Butler MH, Zilberberg N, Gold-stein SA. 2002. Forward transport. 14–3–3binding overcomes retention in endoplasmicreticulum by dibasic signals. Cell 111:577–88

Orci L. 1982. The macro- and micro-domainsin the endocrine pancreas. Diabetes 31:538–65

Orci L, Palmer DJ, Ravazzola M, PerreletA, Amherdt M, Rothman JE. 1993. Bud-ding from Golgi membranes requires thecoatomer complex of non-clathrin coat pro-teins. Nature 362:648–52

Orci L, Ravazzola M, Meda P, Holcomb C,Moore HP, et al. 1991. Mammalian Sec23phomologue is restricted to the endoplasmicreticulum transitional cytoplasm. Proc. Natl.Acad. Sci. USA 88:8611–15

Otte S, Barlowe C. 2002. The Erv41p-Erv46pcomplex: multiple export signals are requiredin trans for COPII-dependent transport fromthe ER. EMBO J. 21:6095–104

Pagano A, Letourneur F, Garcia-Estefania D,Carpentier JL, Orci L, Paccaud JP. 1999.Sec24 proteins and sorting at the endoplas-mic reticulum. J. Biol. Chem. 274:7833–40

Paris S, Beraud-Dufour S, Robineau S, BigayJ, Antonny B, et al. 1997. Role of protein-phospholipid interactions in the activation ofARF1 by the guanine nucleotide exchangefactor Arno. J. Biol. Chem. 272:22221–26

Parlati F, McNew JA, Fukuda R, Miller R,Sollner TH, Rothman JE. 2000. Topologicalrestriction of SNARE-dependent membranefusion. Nature 407:194–98

Patel SK, Indig FE, Olivieri N, Levine ND, Lat-terich M. 1998. Organelle membrane fusion:a novel function for the syntaxin homolog

Ufe1p in ER membrane fusion. Cell 92:611–20

Peng R, Gallwitz D. 2002. Sly1 protein boundto Golgi syntaxin Sed5p allows assembly andcontributes to specificity of SNARE fusioncomplexes. J. Cell Biol. 157:645–55

Peng R, Grabowski R, De Antoni A, GallwitzD. 1999. Specific interaction of the yeastcis-Golgi syntaxin Sed5p and the coat pro-tein complex II component Sec24p of endo-plasmic reticulum-derived transport vesicles.Proc. Natl. Acad. Sci. USA 96:3751–56

Peyroche A, Paris S, Jackson CL. 1996. Nu-cleotide exchange on ARF mediated by yeastGea1 protein. Nature 384:479–81

Poon PP, Cassel D, Spang A, Rotman M, PickE, et al. 1999. Retrograde transport from theyeast Golgi is mediated by two ARF GAPproteins with overlapping function. EMBOJ. 18:555–64

Powers J, Barlowe C. 1998. Transport of Axl2pdepends on Erv14p, an ER-vesicle proteinrelated to the Drosophila cornichon geneproduct. J. Cell Biol. 142:1209–22

Presley JF, Cole NB, Schroer TA, Hirschberg K,Zaal KJ, Lippincott-Schwartz J. 1997. ER-to-Golgi transport visualized in living cells.Nature 389:81–85

Preuss D, Mulholland J, Franzusoff A, SegevN, Botstein D. 1992. Characterization of theSaccharomyces Golgi complex through thecell cycle by immunoelectron microscopy.Mol. Biol. Cell 3:789–803

Reilly BA, Kraynack BA, VanRheenen SM,Waters MG. 2001. Golgi-to-endoplasmicreticulum (ER) retrograde traffic in yeast re-quires Dsl1p, a component of the ER targetsite that interacts with a COPI coat subunit.Mol. Biol. Cell 12:3783–96

Rein U, Andag U, Duden R, Schmitt HD,Spang A. 2002. ARF-GAP-mediated inter-action between the ER-Golgi v-SNAREs andthe COPI coat. J. Cell Biol. 157:395–404

Renault L, Kuhlmann J, Henkel A, WittinghoferA. 2001. Structural basis for guanine nu-cleotide exchange on Ran by the regulatorof chromosome condensation (RCC1). Cell105:245–55

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.



Roberg KJ, Crotwell M, Espenshade P, Gi-meno R, Kaiser CA. 1999. LST1 is a SEC24homologue used for selective export of theplasma membrane ATPase from the endo-plasmic reticulum. J. Cell Biol. 145:659–72

Robineau S, Chabre M, Antonny B. 2000. Bind-ing site of brefeldin A at the interface be-tween the small G protein ADP-ribosylationfactor 1 (ARF1) and the nucleotide-exchangefactor Sec7 domain. Proc. Natl. Acad. Sci.USA 97:9913–18

Rossanese OW, Soderholm J, Bevis BJ, SearsIB, O’Connor J, et al. 1999. Golgi struc-ture correlates with transitional endoplas-mic reticulum organization in Pichia pastorisand Saccharomyces cerevisiae. J. Cell Biol.145:69–81

Sacher M, Barrowman J, Wang W, Horecka J,Zhang Y, et al. 2001. TRAPP I implicatedin the specificity of tethering in ER-to-Golgitransport. Mol. Cell 7:433–42

Sacher M, Jiang Y, Barrowman J, Scarpa A,Burston J, et al. 1998. TRAPP, a highly con-served novel complex on the cis-Golgi thatmediates vesicle docking and fusion. EMBOJ. 17:2494–503

Sandmann T, Herrmann JM, Dengjel J, SchwarzH, Spang A. 2003. Suppression of coatomermutants by a new protein family with COPIand COPII binding motifs in Saccharomycescerevisiae. Mol. Biol. Cell 14:3097–113

Sapperstein SK, Lupashin VV, Schmitt HD,Waters MG. 1996. Assembly of the ER toGolgi SNARE complex requires Uso1p. J.Cell Biol. 132:755–67

Sapperstein SK, Walter DM, Grosvenor AR,Heuser JE, Waters MG. 1995. p115 is a gen-eral vesicular transport factor related to theyeast endoplasmic reticulum to Golgi trans-port factor Uso1p. Proc. Natl. Acad. Sci. USA92:522–26

Sato K, Nakano A. 2003. Oligomerization ofa cargo receptor directs protein sorting intoCOPII-coated transport vesicles. Mol. Biol.Cell 14:3055–63

Sato K, Sato M, Nakano A. 1997. Rer1p as com-mon machinery for the endoplasmic reticu-

lum localization of membrane proteins. Proc.Natl. Acad. Sci. USA 94:9693–98

Sato K, Sato M, Nakano A. 2003. Rer1p, a re-trieval receptor for ER membrane proteins,recognizes transmembrane domains in mul-tiple modes. Mol. Biol. Cell. 14:3605–16

Sato M, Sato K, Nakano A. 1996. Endoplasmicreticulum localization of Sec12p is achievedby two mechanisms: Rer1p-dependent re-trieval that requires the transmembrane do-main and Rer1p-independent retention thatinvolves the cytoplasmic domain. J. CellBiol. 134:279–93

Scales SJ, Pepperkok R, Kreis TE. 1997. Visu-alization of ER-to-Golgi transport in livingcells reveals a sequential mode of action forCOPII and COPI. Cell 90:1137–48

Scheel AA, Pelham HR. 1998. Identificationof amino acids in the binding pocket ofthe human KDEL receptor. J. Biol. Chem.273:2467–72

Schekman R, Orci L. 1996. Coat proteins andvesicle budding. Science 271:1526–33

Schroder-Kohne S, Letourneur F, Riezman H.1998. Alpha-COP can discriminate betweendistinct, functional di-lysine signals in vitroand regulates access into retrograde trans-port. J. Cell Sci. 111:3459–70

Shimoni Y, Kurihara T, Ravazzola M, AmherdtM, Orci L, Schekman R. 2000. Lst1p andSec24p cooperate in sorting of the plasmamembrane ATPase into COPII vesicles inSaccharomyces cerevisiae. J. Cell Biol. 151:973–84

Shorter J, Beard MB, Seemann J, Dirac-Svej-strup AB, Warren G. 2002. Sequential teth-ering of Golgins and catalysis of SNARE-pin assembly by the vesicle-tethering proteinp115. J. Cell Biol. 157:45–62

Siddiqi SA, Gorelick FS, Mahan JT, MansbachCM 2nd. 2003. COPII proteins are requiredfor Golgi fusion but not for endoplasmicreticulum budding of the pre-chylomicrontransport vesicle. J. Cell Sci. 116:415–27

Spang A, Herrmann JM, Hamamoto S, Schek-man R. 2001. The ADP ribosylation factor-nucleotide exchange factors Gea1p andGea2p have overlapping, but not redundant

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.


122 LEE ET AL.

functions in retrograde transport from theGolgi to the endoplasmic reticulum. Mol.Biol. Cell 12:1035–45

Spang A, Schekman R. 1998. Reconstitution ofretrograde transport from the Golgi to the ERin vitro. J. Cell Biol. 143:589–99

Springer S, Schekman R. 1998. Nucleation ofCOPII vesicular coat complex by endoplas-mic reticulum to Golgi vesicle SNAREs. Sci-ence 281:698–700

Springer S, Spang A, Schekman R. 1999. Aprimer on vesicle budding. Cell 97:145–48

Stamnes M. 2002. Regulating the actin cy-toskeleton during vesicular transport. Curr.Opin. Cell Biol. 14:428–33

Stephens DJ, Pepperkok R. 2002. Imag-ing of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgitransport in mammalian cells. J. Cell Sci.115:1149–60

Storrie B, Pepperkok R, Nilsson T. 2000. Break-ing the COPI monopoly on Golgi recycling.Trends Cell Biol. 10:385–91

Supek F, Madden DT, Hamamoto S, Orci L,Schekman R. 2002. Sec16p potentiates theaction of COPII proteins to bud transportvesicles. J. Cell Biol. 158:1029–38

Szafer E, Pick E, Rotman M, Zuck S, HuberI, Cassel D. 2000. Role of coatomer andphospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1. J. Biol. Chem. 275:23615–19

Szafer E, Rotman M, Cassel D. 2001. Regula-tion of GTP hydrolysis on ADP-ribosylationfactor-1 at the Golgi membrane. J. Biol.Chem. 276:47834–39

Taxis C, Vogel F, Wolf DH. 2002. ER-Golgitraffic is a prerequisite for efficient ER degra-dation. Mol. Biol. Cell 13:1806–18

Toonen RF, Verhage M. 2003. Vesicle traffick-ing: pleasure and pain from SM genes. TrendsCell Biol. 13:177–86

Trilla JA, Duran A, Roncero C. 1999. Chs7p,a new protein involved in the control of pro-tein export from the endoplasmic reticulumthat is specifically engaged in the regulation

of chitin synthesis in Saccharomyces cere-visiae. J. Cell Biol. 145:1153–63

Vashist S, Kim W, Belden WJ, Spear ED,Barlowe C, Ng DT. 2001. Distinct retrievaland retention mechanisms are required forthe quality control of endoplasmic reticu-lum protein folding. J. Cell Biol. 155:355–68

Votsmeier C, Gallwitz D. 2001. An acidic se-quence of a putative yeast Golgi membraneprotein binds COPII and facilitates ER ex-port. EMBO J. 20:6742–50

Wang W, Sacher M, Ferro-Novick S. 2000.TRAPP stimulates guanine nucleotide ex-change on Ypt1p. J. Cell Biol. 151:289–96

Watson PJ, Frigerio G, Collins BM, Du-den R, Owen DJ. 2004. gamma-COP ap-pendage domain—structure and function.Traffic 5:79–88

Wen C, Greenwald I. 1999. p24 proteins andquality control of LIN-12 and GLP-1 traffick-ing in Caenorhabditis elegans. J. Cell Biol.145:1165–75

Whyte JR, Munro S. 2002. Vesicle tetheringcomplexes in membrane traffic. J. Cell Sci.115:2627–37

Wieland FT, Gleason ML, Serafini TA, Roth-man JE. 1987. The rate of bulk flow fromthe endoplasmic reticulum to the cell surface.Cell 50:289–300

Wilson DW, Lewis MJ, Pelham HR. 1993. pH-dependent binding of KDEL to its receptorin vitro. J. Biol. Chem. 268:7465–68

Wu WJ, Erickson JW, Lin R, Cerione RA. 2000.The gamma-subunit of the coatomer complexbinds Cdc42 to mediate transformation. Na-ture 405:800–4

Yamaguchi T, Dulubova I, Min SW, Chen X,Rizo J, Sudhof TC. 2002. Sly1 binds to Golgiand ER syntaxins via a conserved N-terminalpeptide motif. Dev. Cell 2:295–305

Yoshihisa T, Barlowe C, Schekman R. 1993.Requirement for a GTPase-activating pro-tein in vesicle budding from the en-doplasmic reticulum. Science 259:1466–68

Yuan H, Michelsen K, Schwappach B. 2003.14-3-3 dimers probe the assembly status of

Ann

u. R

ev. C

ell.

Dev

. Bio

l. 20

04.2

0:87

-123

. Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Col

umbi

a U

nive

rsity

on

06/1

7/05

. For

per

sona

l use

onl

y.



multimeric membrane proteins. Curr. Biol.13:638–46

Zhang B, Cunningham MA, Nichols WC,Bernat JA, Seligsohn U, et al. 2003. Bleed-ing due to disruption of a cargo-specific ER-to-Golgi transport complex. Nat. Genet. 34:220–25

Zhao L, Helms JB, Brugger B, Harter C,Martoglio B, et al. 1997. Direct and GTP-dependent interaction of ADP-ribosylationfactor-1 with coatomer subunit beta. Proc.Natl. Acad. Sci. USA 94:4418–23

Zhao L, Helms JB, Brunner J, Wieland FT.1999. GTP-dependent binding of ADP-ribo-sylation factor to coatomer in close proxim-ity to the binding site for dilysine retrievalmotifs and p23. J. Biol. Chem. 274:14198–203

Zhao X, Lasell TK, Melancon P. 2002.Localization of large ADP-ribosylationfactor-guanine nucleotide exchange factorsto different Golgi compartments: evidencefor distinct functions in protein traffic. Mol.Biol. Cell 13:119–33

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u. R

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ell.

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. Bio

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04.2

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-123

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s.or

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umbi

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06/1

7/05

. For

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l use

onl

y.