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Molecular Biology of the Cell
Sixth Edition!Chapter 12
Intracellular Compartments and Protein Sorting
Hsou-min Li [email protected]!
Copyright © Garland Science 2015
Alberts • Johnson • Lewis • Morgan • Raff • Roberts • Walter!
CHAPTER CONTENTS
The compartmentalization of cells The endoplasmic reticulum (ER) The transport of molecules between the nucleus
and the cytosol The transport of proteins into mitochondria and
chloroplasts Peroxisomes
All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles
THE COMPARTMENTALIZATION OF CELLS
Plant cells have additional unique features: chloroplast (plastid), cell wall and a very large central vacuole
A typical animal cell is between 10 and 100 micro-meters.!
compartment <-> function!
Different cell types have very different abundance and shapes of organelles (membranes)
Liver cells: a lot more mitochondria!
pancreatic cells: a lot more secretory vesicles!
Organelle biogenesis
Why do we have the problem of “protein sorting”?
-- proteins are made at the same place but have to be sorted to different organelles in order to function properly.
Generally divided into: 1. secretory and endocytic
pathways : mostly start with co-translational transport to the ER
2. organelle biogenesis: mostly post-translational
Common mechanism for
protein sorting to all organelles:
1. A targeting sequence 2. A receptor (complex) 3. A translocation channel
across the membrane 4. An energy favorable system
to drive the transport and make the transport uni-directional.
So for all the systems we talk about today, we will ask 4 questions: 1. What is the nature of the targeting signal? (e.g. How does it different from
other signals?) What is the specific sequence motifs? 2. What is the receptor? 3. What is the structure of the channel? (e.g. are the proteins translocated in
folded or unfolded forms?) 4. What is the source of energy? ( i.e. why is the translocation uni-
directional?) ATP? GTP? PMF?
Next !chapter!
secretory and endocytic pathways
The Endoplasmic Reticulum: Introduction
smooth tubules, rough sheets!
ER is structurally dynamic and functionally diverse!1. continuous with outer nuclear envelope!2. Two basic types: rough (sheets), smooth (tubules)!“Rough” example:!
Pancreatic cells need to secrete large amounts of digestive enzymes (proteins!) so they are loaded with rough ER. !
“Smooth” example:!Steroid hormone (lipids!) secreting cells have abundant smooth ER to house the lipid synthesizing and modifying enzymes. !
pancreatic cell!
Steroid hormone secreting cells!
structure
ER network
Rough and smooth ER can be isolated = microsomes
heavier fraction: all from rough ER!
lighter fraction: mixtures of smooth ER, Golgi.....!
The isolated microsomes are powerful tools for studying ER protein transport. For example, from the experiments at left, they found:!1. Transport needs to be co-translational. !2. Proteins become smaller after being
transported into ER – a cleaved signal peptide.!
!
History!
Signal sequences were first discovered in proteins imported into the rough ER!
THE ENDOPLASMIC RETICULUM
• The signal hypothesis
The 1975 signal hypothesis (1999 Nobel Prize), Günter Blobel predicted:!1. an N-terminally localized targeting signal on the protein itself.!2. a binding factor that guides the protein to the ER (deleted in the formal hypothesis,
sigh!)!3. a signal-induced protein-conducting tunnel through the ER membrane.!
1971
1975!
Biomembranes 2 p193-195 (1971)!
The general process of co-translational import into ER
Then we talk about individual components in more details:!SRP, signal sequence, SR, GTP cycle, polysome, Sec61 channel !
Signal-Recognition Particle (SRP): a complex of one RNA and six proteins
blocking the elongation factor binding site!
P54: !1. binds the signal sequence,!2. interacts with SRP receptor,!3. hydrolyzes GTP !
The GTPase switch
1. conformational differences between GTP and GDP states 2. controlled “GTP hydrolysis” and “GDP-to-GTP exchange” by two other proteins
Signal-Recognition Particle (SRP) and SRP receptor (SR) and GTP cycle
Both SRP and SRP receptor are GTPases. They stimulate each other’s GTP hydrolysis and then release each other. Thus GTP ensures the directionality of the SRP cycle. GTP is also used by ribosome for translation.!
empty state ! binding-GTP state ! stimulate each other ! !GDT state-separation ! empty state!
Each mRNA is translated by many ribosomes at the same time -polysome
Each mRNA is translated by many ribosomes at the same time- polysome (polyribosome). Ribosomes are all the same. They form “free” polysome in the cytosol, or “membrane-bound” polysome on ER, depending on which RNA they are translating. !
Schematic representation of membrane-bound polysomes together with microtubules that are linked to the ER membrane via a microtubule-anchoring protein. Two ribosomes that are not part of the polysomes are also shown.
The channel that engages the ribosome-nascent chain complex will form the same pattern!
The Polypeptide Chain Passes Through an Aqueous Channel - the Sec61 complex
16!
Membrane proteins can diffuse laterally into the lipid bilayer through the lateral gate of the channel.!
Identified by yeast mutants (that fail to secret) and confirmed by cross-linking experiments. Composed of three proteins Sec61 α, β, γ, with Sec61α being the major component.!
!How does the channel close: At "closed" state, the channel is sealed
by a plug (formed from one of the α helixes of the Sec61α subunit) and an isoleucine ring at the neck of the channel (a hydrophobic gasket). !
How does the channel open: channel is opened by the nascent chain, which inserts as a loop with the signal peptide intercalated into the lateral gate. !
1975!
JCB 67, p835-851 (1975)!
The picture shows pores formed by 4 trimers (α, β, γ). Cell Vol 87 (4), 1996 !
The 1975 signal hypothesis predicted:!1. an N-terminal localized targeting signal on the
protein itself.!2. a binding factor that guides the protein to ER
(deleted in the formal hypothesis, sigh!)!3. a signal-induced protein-conducting tunnel through
the ER membrane.!
History of the channel opening!1975 pore induced to form by the signal
peptide!1990 ~~ pore always open, sealed by
ribosome and BiP!2004 pore usually closed by a plug, but
doesn’t know how it opens. !2015-16 pore opened by signal peptide!
2004! 2016!
218 The Journal of Cell Biology
|
Volume 156, Number 2, 2002
In This IssueIn This Issue
It’s a chaperone . . . it’s a gatekeeper . . . it’s BiP
magine inserting a piece of string into an inflated balloon without letting any of the air
out. An analogous problem confronts cells in the production of integral membrane-spanning proteins, which must be inserted partway into the ER without letting the ER calcium stores leak into the cytoplasm. On page 261, Haigh and Johnson show that the ER lumenal chaperone protein BiP controls one end of a double door
I
BiP seals the pore.
system at the translocon, the pore through which nascent proteins enter the ER, and propose a model to explain how the cell accomplishes the daunting task of regulating this double door.
On the cytoplasmic side of the ER membrane, the ribosome binds to the translocon to seal it while translocating a nascent polypeptide into the ER lumen. But the ribosome must break this seal to allow a cytoplasmic domain to extend into the cytoplasm. Some mechanism must exist to seal the lumenal side of the translocon when this happens.
Using a clever fluorescence quenching assay in isolated microsomes, the authors found that BiP is required to seal the
Sorting out secretion
roteins that are destined to be secreted from a cell undergo a
multistep sorting and transport process, but until recently a critical portion of the secretory pathway—the transport of cargo from the Golgi apparatus—has been difficult to dissect. On page 271, Harsay and Schekman show that one population
P
lumenal side of the translocon pore at certain stages during the integration of a transmembrane protein, and that BiP can do this even in the absence of other lumenal proteins. This activity of BiP requires ATP hydrolysis, suggesting that the protein may use similar mechanisms in its diverse duties as a chaperone and pore sealer.
The authors propose that translation of a transmembrane sequence causes a BiP-mediated closure of the lumenal side of a translocon, allowing the ribosomal seal to be opened on the cytoplasmic side without breaching the ER membrane. Future studies will focus on identifying the domains of BiP responsible for sealing the translocon, and determining how BiP and the ribosome coordinate their actions across the ER membrane.
!
of yeast secretory proteins apparently takes a detour through an endosomal compartment on its way from the Golgi apparatus to the cell surface. Although some types of mammalian cells appear to use a secretory path-way involving endosomes, the new work is the first demonstration of such a system in yeast, a model system that should help define additional steps in this poorly understood process.
Previous work demonstrated that yeast
sec6
mutants exhibit a post-Golgi secretion defect that causes the accumulation of two populations of secretory vesicles, distinguished by their differing buoyant densities and cargos. In this genetic background, Harsay and Schekman found that mutations in VPS genes, affecting trans-port to an endosomal compartment, also block protein sorting to the high-density secretory vesicles. In these double mutants, proteins normally targeted to the high-density vesicles are instead sorted into the
light-density vesicles.This is the first time newly synthesized
soluble exocytic proteins have been shown to move through an endosomal compartment on the way to being secreted. While it is still unclear why the cell would have two separate secretion pathways, one possibility is that the less abundant high-density vesicles, which are enriched in enzymes for particular metabolic processes, may allow rapid responses to environmental changes without causing membrane expansion.
The cell’s ability to reroute secretory proteins from one pathway to another may also explain why it has been difficult to isolate mutants defective in the post-Golgi portion of the secretion process: both pathways would have to be shut down simultaneously to block secretion. Using mutations that block the high-density vesicle pathway, the authors are now trying to identify genes involved in the light-density vesicle pathway.
!Going in or out? Isolated clathrin vesicles contain outward-bound invertase.
1562iti Page 218 Friday, January 11, 2002 7:45 AM
on February 28, 2008 w
ww
.jcb.orgD
ownloaded from
JCB 2002!
The next Nobel prize on protein trafficking will be:!Schekman and Rothman on vesicle budding and fusion.!
Randy Schekman!2013!
James Rothman!2013!
The role of ER and Golgi in secretion.!!Discovery of ribosome (“Palade granules”), rough ER and the internal structure of mitochondria.!
The “Signal Hypothesis” 1999!
2013
Autophagy!2016!
Translocation Across the ER Membrane Does Not Always Require Ongoing Polypeptide Chain Elongation – post-translational transport into ER
Post-translational translocation: Some proteins are fully translated before being translocated across the ER membrane. Fairly common in yeast (and bacteria secretion across plasma membrane), and occur occasionally in higher eukaryotes. !
Do NOT need: SRP , SR, GTP. A direct interaction between the translocon and the signal sequence appears to be sufficient for targeting to the ER membrane.!
Need: Sec61 complex, Sec63 complex, BiP ATPase and ATP (SecA ATPase in bacteria)!
Sec63 contains a J domain.
Topology (how many time does the protein transverse the membrane and on which side of the membrane are the N and C termini) of all membrane proteins in the secretory pathway is determined during insertion into the ER membrane. When reaching the plasma membrane, cytosolic side remains in the cytosol and the lumenal side becomes the extracellular side. !
*!
*!
*!
*! Topology of an ER membrane protein is determined mostly by three factors:!!1. “positive inside rule” for the first transmembrane domain!!2. how many transmembrane domains the protein have!!3. whether the first transmembrane domain – usually the signal sequence – is cleaved. !
Membrane protein insertion into the ER membrane
1. The positive-inside rule for the first transmembrane domain
+!
N-terminal side more positive!
SRP!
+!
C-terminal side more positive!
SRP!
"Inside" = do not cross membrane!
!Compare the charges on
the two sides of the transmembrane domain. The side that has more positive charges stays on the cytosol and does not cross the membrane. !
2. Use hydropathy plot to predict the number of transmembrane domains!
If you reverse the charge distribution on the two sides of the first transmembrane domain, the orientation of the first transmembrane domain will flip, and other transmembrane domains will usually follow, and the protein topology will be flipped to become N-cytosol , C-lumen.!
+!
3. Is the signal peptide cleaved?
start transfer -> stop transfer -> start transfer........!
ER import summery
ER Tail-anchored Proteins Are Integrated into the ER Membrane by a Special Mechanism
because even when translation is finished, the “signal peptide” (the hydrophobic transmembrane domain) still has not yet emerged from the ribosome. When it emerges, ribosomes are gone!!
!GET mutants: Golgi-ER transport!
e.g. SNARE proteins!see next chapter!
Guided Entry of Tail-anchored Proteins (GET) !
N-linked: Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked oligosaccharide. A block of 14 sugars linked to the NH2 side chain of asparagine. Start to being trimmed immediately after addition.
Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER
Most proteins are fully folded and assembled in the ER. Factors that help folding:!chaperone proteins (for example: BiP (Hsp70 ATPase))!S-S formation through protein disulfide isomerase!glycosylations: N-linked (and O-linked)! (some cytosolic and nuclear proteins are also gycosylated, but only by an single N-acetylglucosamine)!
One copy of this oligosaccharyl transferase sits with each Sec61 translocon.!
Sec61&
see next page for synthesis process
Synthesis of the N-Linked oligosaccharide precursor in the ER membrane
The N-linked Oligosaccharides Are Used as “handles” and “timers” for Protein Folding in the ER
They provide binding sites for calnexin and calreticulin to keep proteins from aggregating so other chaperones can have time to help folding.!
The third glucose is removed and then re-added continuously (like a timer). When proteins stay too long and still can’t be folded, they are translocated out of the ER (see next slide) for degradation by the proteasome system in the cytosol.!
One example:!
Misfolded soluble proteins are recognized by specific proteins (see previous slide), bound by various proteins to prevent aggregation and also to reduce their S-S bonds, and targeted to a translocator, extracted by an ATPase in the cytosol, polyubiquitinated, de-glucosylated and degraded via the proteasome system. Misfolded membrane proteins follow a similar pathway.!
Misfolded Proteins in the ER Activate an Unfolded Protein Response Three examples of known mechanisms: they produce different transcription factors that go into the nucleus to activate the transcription of genes encoding proteins that will assist folding.
UPR signalling mediated by IRE1, PERK and ATF6
(RNA)!
During stress, BiP (the major ER chaperones, an Hsp70) is recruited to bind unfolded proteins!
translation to produce XBP1 protein
Phosphorylation of eIF2 results in inhibited translation of most proteins (so decrease the number of proteins entering ER), but selective translation of some proteins like ATF4
Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor in the ER
Functions of GPI (glycosyl-phosphatidyl-inositol) anchor:!
1. faster diffusion on the plasma membrane!
2. collecting in the lipid raft!3. serving as a signal for polarized
transport (next chapter).!
fatty acid chains!
32!
O-linked: to -OH groups of serine and threonine. One-by-one added from sugar nucleotides in ER and Golgi. Different proteins have different O-linked modifications.!
ER !Golgi.!
ABO blood types (O-linked glycan attached to glycoproteins and glycolipids on the surface of erythrocytes). The presence of absence of glycosyltransferase that add N-acetylgalactosamine or galactose to O-antigen determines a person’s blood type.!
O-linked glycosylation!
Organelle biogenesis
Next !chapter!
secretory and endocytic pathways
THE TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS AND THE CYTOSOL
Basic structure of the nuclear envelope!The outer nuclear membrane is continuous with the ER membrane (both have ribosome attached) and the perinuclear space is continuous with the ER lumen, with similar protein compositions. !!The inner nuclear membrane has distinct protein composition, with lamina being the most well known one. Lamina act as anchoring for chromosomes and cytoplasmic cytoskeleton via protein complexes that span the nuclear envelope. !!Nucleus has large “nuclear pores”.!!
Nuclear Pore Complexes Perforate the Nuclear Envelope
look in from cytosol
look out from inside the nucleus
Nuclear pores are built with different “nucleaoporin” proteins (ring, scaffold, channel....). The disordered region of channel nucleoporins forms a “kelp bed” like mesh in the pore to allow diffusion of small molecules but restrict large molecules, and also to provide docking sites for nuclear import receptors (see below). Even without the help of the nuclear transport machinery, proteins less than 40 kD can diffuse through by themselves. With the machinery, partially assembled ribosomes can be ferried through. !
It is still not known why the nuclear pores have to have such a complicated structure. !
Nuclear Localization Signals Direct Nuclear Proteins to the Nucleus Nuclear localization signals (NLS) are usually short stretches of positively charged amino acids. They can be anywhere in the proteins and they are not processed after reaching inside the nucleus. Proteins without a NLS can bind to another protein with NLS and be transported. NLS can even be coated onto gold particles and results in transport.!
immunofluorescence microscopy!
Gold particles coated with NLS peptides, injected into cells and fixed after various amount of time.!
Nuclear Import Receptors Bind to NLS
Nuclear import receptors (and export receptors) are a family of homologous proteins but each family member has a slightly different binding sequence specificity.!
!Proteins without NLS (“cargo protein 4” in the figure)
can bind to adaptors with NLS and then be imported.!
!Export signals are much less conserved and less
understood. !
37!
The GTPase switch
Ran-GAP is only in cytosol – high concentration of Ran(GDP) in the cytosol!
Ran-GEF is only in nucleus – high concentration of Ran(GTP) in the nucleus.!
1. Nuclear import receptors (“Importins”) bind to nuclear localization signals on the cargo and then bind to the phenylalanine-glycine (FG) repeats of the unstructured domains of the channel nucleoporins.!
Import driven by cargo complex concentration gradient.!
2. Once in the nucleus, Ran(GTP) binds to the receptor in the nucleus and causes cargo release. Ran(GTP)-receptor travel back to cytosol due to concentration gradient. !
3. Once in the cytosol, Ran(GTP) is converted to Ran(GDP) because Ran-GAP is in the cytosol.!
4. Ran(GDP) does not like to bind to import receptor so the receptor is released. !
Nuclear Import: The Ran GTPase imposes directionality on transport through the nuclear pore complexes
high Ran(GTP) concentration
high Ran(GDP) concentration
Import!
Nuclear Export Works Like Nuclear Import, But in Reverse
Ran-GAP is only in cytosol – high concentration of Ran(GDP) in the cytosol!
Ran-GEF is only in nucleus- high concentration of Ran(GTP) in the nucleus.!
!1. In the nucleus, Ran(GTP) binds to the
export receptor (“Exportin”) and promotes cargo binding. Export driven by Exportin-Ran complex gradient. !
2. Once in the cytosol, Ran(GTP) is converted to Ran(GDP). !
3. Ran(GDP) does not like to bind to the export receptor so receptor is released. Ran(GDP) diffuses back into the nucleus.!
4. Empty export receptor binds to nucleoporin and enter the nucleus again. !
Export!
Controlled shuttling of proteins in and out of the nucleus by controlling the exposure of NLS (nuclear localization signal) and NES (nuclear export signal)
Example: Ca2+ concentration and phosphorylation control whether the NLS and NES are exposed: When T cells are activated, intracellular Ca2+ concentration rises. In high [Ca2+], calcineurin binds to NF-AT (nuclear factor of activated T cells) and dephosphorylates it, resulting in exposure of NLS and blocking of NES. NF-AT-calcineurin complex is imported into the nucleus. When the activation shuts off and [Ca2+] drops, calcineurin releases NF-AT, resulting in exposure of NES and re-phosphorylation of NLS.!
Some of the most potent immuno-supressive drugs inhibit the ability of calcineurin to dephosporylate NF-AT.!
THE TRANSPORT OF PROTEINS INTO MITOCHONDRIA AND CHLOROPLASTS - Introduction
Both organelles evolved from endosymbiotic bacteria:!1. They still have their own DNA (so they still perform
transcription and translation within the organelle)!2. Double-membrane envelope,!!so most proteins need to cross two membranes!
! many sub-compartments;!
lumen
Mitochondria typically form extended networks throughout the cytosol, and their overall morphology is controlled by organelle fusion and fission. The ultrastructural organization of mitochondrial membranes varies considerably between mitochondria of different organisms, tissues, and cell types. The diversity of mitochondrial architecture reflecting the diversity of their metabolic functions is apparent when comparing mitochondria from pancreas (A), adrenal cortex (B), astrocytes (C), muscle (D), brown adipose tissue (E), and an Amoeba (F). Not surprisingly, aberrant mitochondrial shapes and ultrastructures are observed in pathological situations. Cell 149, April 27, 2012 DOI 10.1016/j.cell.2012.04.010!
budding yeast. !green: mitochondria. !blue: bud scars!
Mitochondria have different and dynamic morphologies
Freshly isolated tobacco mesophyll protoplasts contain discrete round/oval mitochondria that can be clumped into irregular aggregates. (B) Mitochondria fused into long, tubular structures 4 h after culture. Plant J., 37 (2004), pp. 379-390 !
hsp70!
proteins transported in unfolded forms!!
Mitochondrial targeting signal Amphiphilic alpha helix !
one side hydrophobic: interact with the receptor on the outer membrane!
one side positively charged: for crossing the inner membrane !
hydrophobic side!!
positively charged side!!
1. TO(I)M = Translocon of the Outer (Inner) membrane of Mitochondria 2. Proteins are imported post-translationally 3. Mitochondrial precursor proteins are imported as unfolded polypeptide chains 4. Precursor proteins are imported through contact sites where two membranes are
pressed together 5. energy: (1) ATP hydrolysis by Hsp70, in the cytosol and in the matrix (2) membrane potential. Matrix side negative, thus positively-charged signals
can easily cross.
Basic steps for protein import into mitochondrial matrix
Mitochondrial Protein Import: various specialized complexes for different sub-compartments
SAM (see next page) and OXA complexes are evolved from bacteria (note the direction of transport)!
Mitochondria have specialized complexes for assembly proteins into various subcompartments. !!
Some of the complexes are newly evolved. Some are from bacteria. !
The SAM complex for inserting mitochondrial outer-membrane beta-barrel proteins evolved from the bacterial BAM complex
BAM: beta-barrel assembly machinery SAM: The sorting and assembly machinery
bacteria eukaryotic cell mitochondria
Transport Into the Inner Mitochondrial Membrane and Intermembrane Space Occurs Via Several Routes
inner membrane: at least three different pathways!
intermembrane space: at least two different pathways!
TRANSPORT OF PROTEINS INTO CHLOROPLASTS
Signal: rich in serine, usually about 50-60 amino acids, the longest among organelles.!TO(I)C = Translocon at the Outer (Inner) envelope membrane of Chloroplasts!
1. Proteins are also post-translationally imported. Similar to mitochondria in the need of crossing two membranes at the contact sites, but whether proteins are transported in unfolded or folded forms is not clear yet. !
2. Use ATP and GTP (the two receptors, Toc159 and Toc34, are GTPases), not membrane potential!
Hsp90
Hsp90
3. A two-part signal sequence, one part for crossing the envelope into the stroma and one part for crossing the thylakoid membranes into the thylakoid lumen, direct proteins to the thylakoid lumen inside the chloroplast!
4. At least three pathways for import into the thylakoid – 2 for lumen, 1 for membrane, all derived from bacteria!
One difference: SRP/SR usually for co-translational insertion of very hydrophobic membrane proteins into the inner membrane.!
Two for thylakoid lumen: (1) ΔpH (Tat) for folded
protein, use ΔpH (2) SecA for unfolded
protein (e.g. plastocyanin), use high ATP, SecA and SecY (Sec61 homolog)
At least one for the thylakoid
membrane: SRP/Alb3: for LHCP (the most abundant thylakoid membrane protein)
E. coli (and cyanobacteria) has homologous pathways!!
Peroxisome: Introduction
1. Single membrane. No DNA 2. Peroxisomes use molecular oxygen and hydrogen peroxide to
perform oxidation reactions (marker enzymes: catalase, urate oxidase. There is so much of them that they form crystals).
RH2+O2 ! R+H2O2
H2O2 + R’H2! R’+2H2O (e.g. oxidation of the ethanol we drink) 2H2O2 ! 2H2O + O2 (by catalase)
3. Another major function: breaking down fatty acids through beta oxidation to make acetyl CoA
4. animal peroxisome: synthesize plasmalogen (the first two steps are in peroxisomes) – the most abundant phospholipids in myelin – why peroxisomal disorders lead to neurological disease
5. Plant peroxisome – a subclass called glyoxysome, which can take two acetyl CoA to make one succinic acid – turns fatty acids into carbohydrates.
E. Newcomb!
6. Two types of signals PTS1: for most matrix proteins. C-terminal tripeptide serine-lysine-leucine-
COOH or a conserved variant. PTS2: near N terminal, but not necessarily on the N terminus. (R/K)(L/V/I/
Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F) Processed in mammal and higher plants, but not in yeast.
Peroxisomes proliferate through two mechanisms: de novo biogenesis through precursor vesicles budding off from ER, and fission of pre-existing peroxisome
1. some membrane proteins insert into ER membranes directly. Some of these serve as receptors for matrix protein import. !
2. PTS1 and PTS2 pathways converge at the membrane channel.!
3. Peroxisomes import proteins in folded forms (gold particles coated with PTS1 can be imported. See next slide). Receptor recycling requires mono-ubiquitination of the receptor and ATP.!
4. Receptor poly-ubiquitination results in receptor degradation. !
Pex19 is required for vesicle budding from ER!
1!
3!
4!2!
Proteins are guided to their destined compartment through specific pathways (previous slides). No matter which pathway, they are all guided by sorting signals in their amino acid sequence that function either as signal sequences or signal patches. Sorting signals are then recognized by specific receptors.
(KDEL)
(SKL)
