Secretory Pathway Flashcards
(133 cards)
1
Q
What pathway did George Palade define using EM and membrane fractionation in the 1950s-1960s?
A
The secretory pathway:
(1) ER -> Golgi
(2) Golgi -> Secretory granule
(3) Secretory granule -> Cell surface
2
Q
What isotopes are used in autoradiography and why?
A
Tritium (³H) is used because it emits a low-energy beta particle that travels only a short distance.
3
Q
How is a sample prepared for autoradiography?
A
- Label sample with tritium-tagged amino acids.
- Fix and embed sample in Epon for EM.
- Section the sample for microscopy.
4
Q
How does autoradiography work after sample preparation?
A
- Place a photo-sensitive silver emulsion over the sample.
- Store in the dark at low temperature → allows radioactivity to expose the emulsion.
- Treat with developer → produces metallic silver from exposed emulsion.
- Grains of metallic silver are visible under electron microscopy.
5
Q
What technique provided the first direct evidence for the secretory pathway?
A
Pulse-chase using autoradiography and electron microscopy showed that secreted proteins appear first in the ER, then in the Golgi.
6
Q
What are the major organelles of the secretory pathway and their functions?
A
(1) ER
- Insertion of newly synthesized proteins.
- Folding
- N-linked glycosylation
- Quality control
(2) Golgi apparatus
- Modification of N-linked oligosaccharides
- Sorting to various destinations (cell surface, secretory granules, endosomes)
7
Q
What are the categories of protein transport between compartments?
A
- Transmembrane transport - Cytoplasm to: ER, mitochondria, peroxisome, chloroplast.
- Gated transport - Through nuclear pores.
- Vesicular transport - Between organelles of:
Secretory pathway (ER -> Cell Surface (inside to outside))
Endocytic pathway (Cell surface -> Lysosomes (outside to inside))
(vesicle leaves on organelle -> fuses with another) - Direct connections - “kiss-and-run” in endocytic pathway, between Golgi stacks.
- Diffusion through cytoplasm - For peripheral proteins on the outside of a compartment.
8
Q
What are the steps in vesicular trafficking?
A
- sorting of cargo, budding, and separation from source membrane
- transfer to destination
- storage (in some cases, e.g. synaptic
vesicles) - recognition of target membrane and fusion
9
Q
What are the three major coat proteins involved in vesicle transport, and where are they found?
A
1) COPII – Found only on the ER (involved in ER exit).
2) COPI – Found on the Golgi (involved in Golgi to ER trafficking).
3) Clathrin – Found on the cell surface, Golgi, and endosomes (involved in multiple types of vesicle formation).
10
Q
What are the steps of vesicle formation?
A
- Sorting of cargo – Coat proteins bind to the cytoplasmic domain of cargo proteins, concentrating them in the vesicle.
- Budding – Vesicle buds off from the source membrane.
- Coat shedding – Coat proteins are lost after budding, allowing the vesicle to move freely and fuse with the target membrane.
11
Q
What is the function of coat proteins in vesicle formation?
A
Coat proteins bind to the cytoplasmic domain of cargo proteins → concentrate them in the vesicle → facilitate budding from the source membrane → shed after budding to allow fusion with the target membrane.
12
Q
What are tubular transport intermediates?
A
Tubular transport intermediates are elongated membrane structures that help in vesicle transport between organelles.
13
Q
Why was clathrin the first coat protein discovered?
A
Clathrin was highly visible under electron microscopy and was discovered in the early 1960s during studies of lipoprotein particle binding and vesicle formation.
14
Q
What is the function of clathrin-coated vesicles?
A
- Form at the cell surface, Golgi, and endosomes.
- Recognize tyrosine and dileucine signals.
- Use Arf1 GTPase.
- Handle multiple types of vesicle formation.
15
Q
What is the function of COPI-coated vesicles?
A
- Found on the Golgi and pre-Golgi.
- Recognize dilysine signals.
- Involved in Golgi to ER trafficking.
- Use Arf1 GTPase.
- Not found on endosomes.
16
Q
What is the function of COPII-coated vesicles?
A
- Found only on the ER.
- Involved in vesicle formation for ER exit.
- Recognize di-acidic and diphenylalanine motifs.
- Use Sar1 GTPase.
17
Q
What is the function of caveolin-coated vesicles?
A
- form at the cell surface
- involved in transport within cholesterol-rich raft domains.
18
Q
What is the function of retromer-coated vesicles?
A
- Found on endosomes.
- Involved in recycling cargo receptors to the Golgi.
19
Q
What are the major motor proteins involved in vesicle transport and what do they bind to?
A
(1) Dynein – Moves toward the (-) end of microtubules (toward the cell center in fibroblasts).
(2) Kinesin – Usually moves toward the (+) end of microtubules (toward the cell periphery in fibroblasts).
(3) Myosin – Moves along actin filaments (usually for short-distance transport).
20
Q
What cytoskeletal element do dynein and kinesin use for transport?
A
Microtubules – Dynein moves toward the (-) end (cell center), and kinesin moves toward the (+) end (cell periphery).
21
Q
What cytoskeletal element does myosin use for transport?
A
Actin filaments – Myosin is usually involved in short-distance transport.
22
Q
In fibroblasts, which motor protein moves vesicles toward the cell center and which moves toward the cell periphery?
A
- Dynein → Cell center (toward (-) end of microtubules).
- Kinesin → Cell periphery (toward (+) end of microtubules).
23
Q
Are there any kinesins that move toward the minus end of microtubules?
A
Yes, but most kinesins move toward the plus end of microtubules.
24
Q
What proteins are responsible for the initial recognition of the target membrane during vesicle docking?
A
Tethering proteins (docking factors) and Rabs.
25
What proteins are responsible for vesicle fusion with the target membrane?
v-SNARE/t-SNARE interactions.
26
What is the role of tethering proteins and Rabs in vesicle transport?
They are involved in the initial recognition of the target membrane.
27
How do v-SNARE and t-SNARE proteins contribute to membrane fusion?
- v-SNARE – Located on the vesicle membrane.
- t-SNARE – Located on the target membrane.
- When they interact, they pull the membranes together to drive fusion.
28
Why are organelle-specific SNAREs important in vesicle transport?
Different SNAREs are specific to different organelles, contributing to accurate targeting and fusion.
29
What type of vesicle is responsible for transport from the ER to the Golgi?
COPII vesicles
30
What types of signals can COPII recognize on the cytoplasmic domains of proteins?
1. Two phenylalanines at the C-terminus.
2. A cluster of acidic amino acids (cytoplasmic, but not necessarily at the end).
31
What happens after COPII vesicles bud off from the ER?
1. COPII vesicle uncoating – Coat is lost after budding.
2. Fusion of COPII vesicles → Forms vesicular-tubular intermediate (VTC) or pre-Golgi intermediate.
3. Pre-Golgi intermediate moves to Golgi using dynein and microtubules.
4. Pre-Golgi intermediate fuses with the Golgi.
32
What motor protein is involved in transporting the pre-Golgi intermediate to the Golgi?
Dynein - Moves toward (-) end of microtubules (toward the cell center).
33
What happens to the COPII coat after budding?
The COPII coat is lost after the vesicle buds off from the ER.
34
How does COPII form a vesicle?
- COPII subunits are found in the cytoplasm.
- They must be brought to ER membranes to assemble and form a coat.
- They must then be released after vesicle budding is complete.
35
What are the two subunits of COPII coats that you need to memorize?
Sec13/31
Sec23/24
36
What small GTPase is involved in COPII coat formation?
Sar-1
37
How does Sar-1 regulate COPII coat formation?
1. GDP-bound Sar-1 → Inactive, in cytoplasm.
2. GTP-bound Sar-1 → Active, binds to the membrane and recruits coat proteins.
38
What happens when Sar-1 is bound to the membrane with GTP?
1. Sar-1 recruits Sec13/31 and Sec23/24.
2. Sec23/24 binds membrane signals and proteins.
3. Sec13/31 binds Sec23/24 → Drives vesicle formation.
4. Sec13/31 causes a conformational change in Sec23/24 → Triggers Sar-1 to hydrolyze GTP → Converts to GDP.
5. Sar-1 comes off → Destabilizes the coat → Coat comes off.
39
What protein acts as the GEF (guanine exchange factor) for Sar-1?
Sec12
40
What happens after the COPII coat is lost?
The vesicle uses tethering factors to fuse with the target membrane.
41
What is the role of Sar1 in COPII formation?
1. Sar1-GDP = Inactive, in cytoplasm.
2. Sar1-GDP is recruited to the membrane by Sec12 (GEF).
3. Sec12 loads Sar1 with GTP → Activates Sar1.
4. Sar1-GTP recruits inner COPII subunit (Sec23/24) → Cargo recruitment.
5. Sec23/24 binds outer COPII subunit (Sec13/31) → Membrane curvature → Budding.
6. Sar1 hydrolyzes GTP → Converts to GDP → Sar1 detaches.
7. Without Sar1, COPII coat detaches → Uncoating of vesicle.
42
What are the roles of the COPII subunits?
- Sar1 – Small GTPase, controls coat formation and uncoating.
- Inner COPII subunit (Sec23/24) – Recognizes cargo.
- Outer COPII subunit (Sec13/31) – Drives budding by forcing membrane curvature
43
What happens after COPII uncoating?
1. Uncoated vesicles are clustered by the golgin tethering factor p115.
2. Rab1-GTP recruits p115 to vesicles.
3. Vesicles tether and fuse using NSF and SNAREs.
4. Correct v-SNARE and t-SNARE interaction = Fusion → Forms vesicular tubular cluster (VTC).
44
How are soluble proteins (like pancreatic enzymes) recruited into COPII vesicles if they don’t have transmembrane domains?
- Cargo receptors (transmembrane proteins) bind soluble proteins and recruit them into COPII vesicles.
- Some proteins may be accidentally trapped during vesicle formation
45
What is the order of COPII vesicle formation and fusion?
1. Sar1-GDP → Sar1-GTP by Sec12.
2. Sar1-GTP recruits Sec23/24 (inner) → Cargo selection.
3. Sec23/24 recruits Sec13/31 (outer) → Budding.
4. Sar1 hydrolyzes GTP → Uncoating.
5. Rab1-GTP recruits p115 → Vesicle tethering.
6. SNAREs interact → Vesicle fusion → VTC formation.
46
How do ER proteins without transmembrane domains get into COPII vesicles?
- They bind to cargo receptors (transmembrane proteins) that can interact with COPII.
- Example: ERGIC53 – Binds to N-linked oligosaccharides on soluble proteins.
47
What happens to cargo receptors after delivering cargo to the Golgi?
- They must be recycled back to the ER using a retrograde pathway.
- Retrograde transport uses a different coat protein: COPI.
48
How do cargo receptors like ERGIC53 interact with COPI and COPII?
(1) COPI interaction → Two lysines at the C-terminus.
- Example: ERGIC53 ends in -KKFF → KK = COPI signal.
(2) COPII interaction → Two phenylalanines (FF) at the C-terminus.
- FF = Strong COPII-binding signal.
49
What is a summary of cargo receptor function?
1. ER protein binds to cargo receptor (e.g., ERGIC53).
2. Cargo receptor binds COPII → Vesicle formation → Transport to Golgi.
3. Cargo receptor releases cargo at Golgi.
4. Cargo receptor binds COPI → Returns to ER for reuse.
50
What are the names for Pre-Golgi Intermediates?
(1) VTC = Vesicular-Tubular Complex
(2) Pre-Golgi Intermediate
(3) IC = Intermediate Compartment
51
How are Pre-Golgi Intermediates formed?
1. Fusion of many COPII vesicles → Forms a tubular complex.
2. Pre-Golgi intermediates are often coated with COPI at the tips.
52
What is the role of Arf1 in COPI vesicle formation?
1. Arf1 = Small GTPase (like Sar1 for COPII)
2. Arf1-GTP on membrane → Recruits COPI coat proteins → Vesicle budding.
53
What happens to cargo receptors after delivering cargo?
1. COPI vesicles bud off from the pre-Golgi intermediate.
2. COPI vesicles return empty cargo receptors and other retrograde cargo to the ER.
54
How do pre-Golgi intermediates move to the Golgi?
1. Dynein → Moves toward the (-) end of microtubules (toward the nucleus).
2. Kinesins (usually) → Move toward the (+) end of microtubules (toward the cell periphery).
3. ER to Golgi movement = Dynein-dependent since it’s toward the nucleus.
55
What are the three steps in vesicle fusion with the cis-Golgi?
(1) Tethering:
Tethering proteins = Golgins (e.g., p115)
p115 binds to Rab1 (small GTPase) → Essential for docking
(2) SNARE interaction
V-SNARE → On vesicle membrane
T-SNARE → On target membrane (cis-Golgi)
Correct pairing = Strong binding → Stabilizes membranes
(2) Membrane Fusion
- VTCs dock with cis-Golgi → Fusion occurs
- Interaction between Rab1–p115 complex (on VTC) and GM130–GRASP65 (on cis-Golgi) facilitates this
- p115 is critical → Blocking it = Major disruption
- GM130 is less critical → Blocking it = No big issue (other alternatives available)
56
Why do proteins need to be recycled from the Golgi back to the ER?
Proteins like cargo receptors and v-SNAREs need to be recycled for reuse.
57
What is the reverse pathway from the Golgi to the ER called?
Retrograde trafficking
58
What are the two types of Golgi-to-ER retrograde pathways?
1. COPI-dependent pathway:
Primary pathway for recycling proteins from the Golgi to the ER.
Involves COPI-coated vesicles.
2. COPI-independent pathway:
Specialized pathway that does not require COPI.
Involves the formation of tubular transport intermediates.
Primarily used for lipid recycling.
59
What type of vesicles are used in COPI-dependent retrograde trafficking?
COPI-coated vesicles
60
What does COPI-independent retrograde trafficking involve?
Tubular transport intermediates (no COPI involved)
61
What are some characteristics of COPI?
- Exact role controversial
- Binds dilysine (-KKXX) motifs found on
ER/Golgi resident proteins
- Responsible for VTC to ER and also Golgi
to ER trafficking
- MAY also be involved in trafficking inside
the Golgi (to be discussed in a later lecture)
62
What are the two major centrifugation techniques used for separating cellular components?
1) Differential centrifugation - Separates particles based on size and mass. Heavier particles like nuclei sediment faster. Timing is important because everything sinks to the bottom.
2) Density gradient centrifugation – Separates particles based on buoyant density using a sucrose or metrizamide gradient. Particles stop moving once they reach a point where centrifugal force = buoyant force. Timing is not important since particles settle at their equilibrium point.
63
How did Schekman use density gradient centrifugation to isolate sec mutants?
He wanted to separate heavy yeast (accumulating proteins due to secretion failure) from light yeast. He exposed yeast to a mutagen, grew them at 24°C (permissive temperature), then shifted them to 37°C (non-permissive). Yeast that couldn’t secrete proteins became heavier and were isolated using density gradient centrifugation.
64
How did Schekman identify sec mutants after centrifugation?
After centrifugation, yeast was shifted back to 24°C to allow survival. Colonies were screened, and mutations were identified in 23 different genes using complementation analysis.
65
What is differential centrifugation and how does it work?
Involves spinning a suspension of cellular components. Larger and heavier components (like nuclei) sediment faster and form a pellet at the bottom. Separation depends on the sedimentation coefficient, which is based on size and shape.
66
What is density gradient centrifugation and how does it work?
A sucrose or metrizamide gradient is created with higher density at the bottom of the tube. Particles move until their buoyant density matches the gradient density — independent of size and shape.
67
How did Schekman create temperature-sensitive mutants for secretion failure?
1) Exposed yeast to a mutation.
2) Grew at 24°C → No secretion block.
3) Shifted to 37°C → Mutants that couldn’t secrete became heavier.
4) Centrifuged to isolate heavier mutants.
5) Shifted back to 24°C to allow survival.
68
Why did Schekman use temperature-sensitive mutations?
At 37°C, secretion is blocked, proteins accumulate, and yeast get heavier — allowing for separation. After cooling back to 24°C, yeast can survive if secretion is restored.
69
How did Schekman determine that 23 genes were involved in secretion?
Used complementation analysis:
- Yeast can be haploid or diploid.
- Haploid yeast → Single gene copy → If mutated, no backup.
- Mated mutant yeast to form diploids.
- If diploid yeast survived at 37°C → mutations in different genes.
- If diploid yeast failed at 37°C → mutations in same gene → Placed in same complementation group.
70
What are complementation groups and how do they work?
- Haploid yeast → One gene copy → Mutation = loss of function.
- Diploid yeast → Two copies → If one copy is WT → Normal function (recessive mutation).
- If two different sec mutants were mated → Survived = Different genes.
- If two sec mutants failed at 37°C → Same gene = Same complementation group.
71
How were sec mutants characterized after isolation?
- Electron Microscopy
- Sorted into groups based on where proteins accumulated
- ER accumulation → Block in ER exit.
- Golgi accumulation → Enlarged Golgi ("Berkeley bodies").
- Vesicle accumulation → Defect in vesicle trafficking.
72
What are the key COPII subunits involved in ER accumulation?
Sec12
Sec13
Sec23
73
How did Schekman’s lab isolate (discover) COPII vesicles?
1. Incubated ER membranes with cytosol, ATP, and GTP.
2. Added a non-hydrolyzable GTP analog → vesicle formation blocked at a specific stage.
3. Isolated COPII vesicles.
74
What sec proteins were identified in COPII vesicles?
Sar1
Sec23
Sec24
Sec13
Sec31
75
What is the limit of resolution (d)?
It is the smallest distance between two points that can still be distinguished.
76
How does near-field microscopy work? What was the limitation of near-field microscopy?
An optical fiber with a 30 nm tip is moved slowly over the sample, evading the diffraction limit to enable superresolution fluorescence imaging.
Image acquisition was slow (30 min per image), making it impractical for biological use, though fine for materials science.
77
How does TIRF microscopy work? What part of the sample is observed in TIRF microscopy? What is the main advantage of TIRF microscopy?
It uses wide-field fluorescence microscopy with illumination at a very shallow angle, creating an evanescent wave that reflects off the coverslip surface, with minimal penetration into the sample.
Only the portion very close to the coverslip can be observed.
It has very low background noise since much of the sample (potentially autofluorescent) is not visible, which is ideal for observing faint fluorescence from single molecules.
78
How does STORM work?
The sample is placed in a special buffer that drives most fluorescent dye molecules into a dark state. The remaining molecules are distant and can be individually visualized as they randomly flicker in and out of the dark state. Images are taken at about 10 images per second for approximately 15 minutes. It allows the visualization of molecules with a localization of ~30 nm.
79
How does PALM work?
Similar to STORM, but uses special photoactivatable GFPs (paGFPs). A small proportion of paGFP molecules can be turned on by near-ultraviolet light, imaged, and then bleached, with this process repeated about 1000 times. It allows for point localization and results similar to STORM.
80
What are the limitations of PALM and STORM?
- Not compatible with confocal microscopy (due to dim images).
- Best for thin samples, as thicker samples may result in background autofluorescence overwhelming the faint signal.
- Observation limited to the bottom of the sample when using TIRF.
- Often used on thin sections prepared for electron microscopy.
- Difficult to perform with living cells (though not impossible).
81
How is the Golgi organized, and what happens to cargo inside it?
The Golgi resembles a stack of pancakes and is polar. Cargo enters at the cis face and exits at the trans face (sometimes referred to as the trans Golgi network). Inside the Golgi, cargoes can be modified by enzymes, and its structure helps compartmentalize these modification functions.
82
What are some functions of the Golgi?
- Sequential modifications of N-linked
oligosaccharides
- O-linked glycosylation
- Proteoglycan glycosylation
- Proteolytic modifications of proteins (late Golgi)
- Sorting of proteins to various destinations (late
Golgi)
- Production of secretory granules (late Golgi)
83
What is Glycosylation?
Glycosylation is the reaction in which a carbohydrate, (i.e. a glycosyl donor) is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other molecules.
84
How are glycosylation types classified?
according to the
identity of the atom of the amino acid which
binds the carbohydrate chain, i.e. C-linked, Nlinked or O-linked.
85
Where does C-glycosylation, N-glycosylation, and O-glycosylation take place?
C-glycosylation and initial N-glycosylation take
place in the endoplasmic reticulum.
Modifications of N-glycosylations take place in both the endoplasmic reticulum and the Golgi apparatus.
In contrast, most O-glycosylation takes place
entirely in the Golgi apparatus.
86
What happens during the sequential modifications of N-linked oligosaccharides?
- Oligosaccharides attach to Asparagine and initially have a high-mannose structure, containing mannose and glucose. These are involved in ER quality control signals.
- The structure is trimmed once the process is complete.
- The oligosaccharides become resistant to removal by Endo-H after modification.
87
Where do the enzymatic modifications of N-linked oligosaccharides occur, and how do they work?
- The steps of N-linked oligosaccharide modification occur in different parts of the Golgi lumen, primarily in the medial Golgi.
- Enzymes are efficient when they are in the right order, which is how the Golgi processes them.
88
Why is glycosylation important for protein folding, and what happens if it is blocked?
- Glycosylation is crucial for protein folding—proteins won't fold properly without oligosaccharides.
- In a cell line, blocking glycosylation may lead to cell death, but some steps can be blocked in tissue culture and still allow the cell to survive.
89
How can glycosylation provide insight into neighboring cells?
Glycosylation patterns can help identify neighboring cells and are important for processes like development.
90
Where does O-linked glycosylation occur and what is added to the protein?
- O-linked glycosylation occurs in the medial Golgi apparatus.
- N-acetyl-galactosamine is linked to serine (often just before glycine) or to threonine.
- Additional sugars may then be added, and in proteoglycans, this process can continue until a chain of 100 or more repeating saccharide units is formed.
91
How are O-linked glycosylation chains further modified and what is their function?
- These chains can be further modified by sulphation in the Golgi.
- Secreted proteoglycans form a large portion of the extracellular matrix.
92
What is the difference between N-linked and O-linked glycosylation in terms of their cellular roles?
- N-linked glycosylation happens on almost every protein that leaves the ER. It may act as a signal for proteins to leave the ER.
- O-linked glycosylation occurs on only some proteins and is exemplified in proteoglycans.
93
What is the pH of the trans-Golgi network (TGN) and how does it vary?
The TGN is slightly acidic, with a pH around 6.0 (this can vary by cell type).
94
What types of proteases are found in the TGN, and what do they do?
- Acidic proteases, such as furin, are found in the TGN.
- These proteases process secreted or lysosomal proteins into their mature/active forms.
95
How does the acidic pH in the TGN affect protein aggregation?
- The acidic pH in the TGN drives the aggregation of some secreted proteins, such as pancreatic digestive enzymes.
- These proteins are incorporated into secretory granules at high density.
- A further drop in pH in the secretory granules (e.g., pancreatic condensing vacuoles) leads to further aggregation and compaction of proteins into the smallest possible volume for secretion.
96
What is the structure and organization of the Golgi apparatus?
1) The Golgi is polar, with a cis (entry) face and trans (exit) face.
2) It consists of stacks of layered cisternae, resembling a stack of pancakes.
3) Golgi enzymes, especially those modifying N-linked oligosaccharides, are localized to specific regions of the Golgi.
97
How are Golgi stacks arranged?
- Golgi stacks are arranged in long ribbons, with disorganized areas between stacks.
98
What were the Intra-Golgi trafficking theories in the 1970s?
1. Proteins may diffuse through connections between cisternae.
2. The cis cisterna may gradually mature to become the trans cisterna.
3. Each cisterna could be a separate compartment, requiring vesicles to transport cargo.
99
What evidence supported the cisternal maturation theory in the Golgi?
- Electron microscopists observed that trans cisternae seemed to be “peeling off” and disassembling, which favored the cisternal maturation theory.
- However, this theory could not explain how the Golgi enzymes were segregated into different cisternae.
100
What is COPI and what role does it play in intracellular transport?
1. COPI is found on VTCs (Vesicular-Tubular Clusters) and Golgi rims, particularly on the cis-Golgi.
2. It is responsible for retrograde transport from the Golgi/VTC to the ER, including the transport of cargo receptors like the KDEL receptor.
3. COPI may also be involved in retrograde intra-Golgi transport.
101
What is the composition of COPI?
- COPI is composed of 7 polypeptide chains.
- These chains assemble into a single permanent structure, unlike the layered subunits seen in COPII and clathrin coats.
- subunits share homology with AP1 (Adaptor protein 1)
- These subunits bind to the small GTPase Arf1, which plays a crucial role in the COPI coat function.
102
What is the function of COPI subunits in membrane curvature and cargo recognition?
- α, β', and ε subunits are involved in cage formation, which likely drives membrane curvature, corresponding to functions similar to outer subunits in COPII or clathrin.
- Outer COPI subunits recognize KK motifs on cargo, assisting in the binding and recognition of transport targets.
103
What is the mechanism of Arf1 and COPI function in vesicle formation?
1. Arf1-GDP in the cytoplasm is loaded onto the membrane by GEF (GBF1), which exchanges GDP for GTP.
2. Arf1-GTP recruits COPI, which in turn recruits cargo.
3. COPI induces membrane curvature and vesicle budding.
4. ArfGAP1/ArfGAP2 (not clearly defined) binds to COPI vesicles, causing Arf1-GTP to hydrolyze its GTP into GDP. Arf1-GDP cannot remain on membranes.
5. Once Arf1-GDP dissociates, the COPI coat leaves the membrane, uncoating the vesicle.
104
How does the regulation of Arf1 and vesicle formation differ in clathrin-coated vesicles?
- The regulation of Arf1 in clathrin-coated vesicles is more complex and poorly understood.
- Clathrin vesicle formation also involves Arf1, but the exchange factor and GAP proteins involved can differ from those used in COPI vesicles.
105
What are the classes of cargo receptors in the early secretory pathway, and what are their functions?
(1) KDEL Receptor:
Binds escaped proteins in the Golgi and returns them to the ER.
Works in reverse compared to most cargo receptors.
Binds KDEL at acidic pH (in Golgi/VTC) and releases at neutral pH (in the ER).
(2) ERGIC53:
Binds cargo with high-mannose oligosaccharides in the ER.
Releases cargo in the slightly acidic environment of VTCs (likely around pH 6).
(3) P24 Family:
Binds GPI-anchored proteins.
Binds at neutral pH (ER) and releases at mildly acidic pH (in ERGIC).
(4) Surf4:
Binds a three-amino acid sequence at the N-terminus of some soluble cargo proteins.
106
How do most cargo receptors facilitate the movement of cargo?
(1) Exit from ER:
Cargo receptors have sequences (like FF at the carboxy terminus) that interact with COPII proteins, allowing them to exit the ER.
(2) Return to ER:
Cargo receptors have sequences (like KK or its variations) that interact with COPI proteins, allowing them to return to the ER from the VTC or Golgi.
107
How does pH sensitivity affect cargo receptor binding and release?
(1) Neutral pH (in ER): Cargo receptors bind cargo.
(2) Slightly acidic pH (in Golgi or VTC): Cargo receptors lose binding with cargo and release it.
108
What is the role of the KDEL sequence in ER retention?
- KDEL sequences are found on resident ER proteins that should remain in the ER.
- These proteins are not efficiently recruited to COPII vesicles but can accidentally leave the ER.
- The KDEL receptor in the Golgi binds the KDEL sequence and returns the proteins to the ER via COPI vesicles.
109
What is the specific binding behavior of the KDEL receptor?
1. Binds KDEL at acidic pH (in the Golgi/VTC).
2. Releases KDEL at neutral pH (in the ER).
This is the reverse specificity of the forward cargo receptors.
110
What was James Rothman's hypothesis about cargo transport through the Golgi apparatus?
Rothman hypothesized that cargo was transported between cisternae by coated vesicles.
111
What was the goal of Rothman’s in vitro system for studying Golgi trafficking?
The goal was to analyze cargo transport between two populations of Golgi fractions using coated vesicles.
112
What techniques were used for membrane fractionation to purify Golgi fractions?
Differential Centrifugation, Density Gradient Centrifugation, and Multiple Fractionation Steps.
113
What was the challenge Rothman faced in membrane fractionation?
The challenge was to purify Golgi fractions while keeping them functional for cargo transport studies.
114
What is a cell-free system, and why is it used in studying Golgi transport?
A cell-free system isolates the Golgi process from a full cell, allowing researchers to focus on the minimal components required for cargo transport.
115
What are the key components of a cell-free system?
Cytosol, ATP, GTP, and Golgi fractions.
116
How did Rothman set up his in vitro experiment for intra-Golgi trafficking?
Rothman used two populations of CHO cells:
One with VSV G protein but lacking GlcNAc transferase.
The other with GlcNAc transferase but lacking VSV G protein.
These populations were mixed, and cytosol + ATP were added.
117
How was GlcNAc incorporation detected in the experiment?
VSV-G was immunoprecipitated and GlcNAc incorporation was measured using SDS-PAGE and radioactive scintillation.
118
What observation was made using electron microscopy in Rothman’s experiment?
Coated pits and coated vesicles were visible on the Golgi during in vitro transport reactions.
119
What hypothesis did Rothman propose about vesicle formation and fusion?
Rothman hypothesized that a sequence of events was required to form vesicles and fuse them with target membranes.
120
How did Rothman identify the COPI coat in his experiments?
Rothman added a non-hydrolyzable GTP analog (GTPgS), which caused vesicles to become coated. After purification, peptides were identified and named COP proteins.
121
What small protein was discovered to be essential for COPI vesicle formation?
Arf, a small GTPase, was identified as essential for COPI vesicle formation.
122
What was one major accomplishment of Rothman’s in vitro system?
The identification of the COPI coat and the key cytosolic proteins required for intra-Golgi transport.
123
What process did Rothman’s cell-free system help elucidate?
The minimal components required for vesicle trafficking and cargo transport through the Golgi apparatus.
124
What key observation supported the idea of cisternal maturation in algal cells?
Algal scales were found in specific Golgi cisternae at different stages, suggesting that cisternae mature and progress through the Golgi rather than cargo being shuttled between static cisternae.
125
What observation in mammalian cells provided direct evidence for cisternal maturation?
Procollagen was seen to appear first in the cis-Golgi, then in the medial compartments, and finally in the trans-Golgi — it was not spread across the entire Golgi.
125
Why was procollagen a good model for testing cisternal maturation?
Procollagen only exits the ER in the presence of ascorbic acid, allowing researchers to track its movement through the Golgi over time.
126
How did the ts045 VSVG mutant provide evidence for cisternal maturation?
1. VSVG misfolds at 40°C and is retained in the ER by quality control.
2. When the temperature drops to 35°C, it folds correctly and exits the ER.
3. Once out of the ER, it follows the secretory pathway, moving through the Golgi in a manner consistent with cisternal maturation.
127
If cisternal maturation explains cargo movement, what problem remains?
Golgi enzymes (like Mannosidase II) are always in the correct location despite the cisternae moving. This suggests that the enzymes must be actively relocated.
128
What are the two possible models for how Golgi enzymes remain in place during cisternal maturation?
1. COPI vesicles move enzymes backwards at the same rate as cisternal maturation.
2. Enzymes diffuse within the Golgi and are localized by affinity for their substrates (evidence for this is lacking).
129
Why is the COPI vesicle model more likely than free diffusion?
(1) There are different variants of COPI vesicles, but it’s unclear how they are fine-tuned.
(2) Resident proteins have shorter transmembrane domains than cargo proteins, so they might localize to different lipid domains.
130
What is COPI’s known role in the secretory pathway?
COPI is required for recycling proteins from the Golgi and VTC (vesicular-tubular clusters) back to the ER.
131
What evidence supports COPI’s role in recycling to the ER?
1. Cargo receptors are highly concentrated in COPI vesicles on both the Golgi and VTC.
2. Cargo receptors have COPI-interacting sequences on their cytoplasmic side.
3. Deletion of COPI subunits in yeast causes cargo receptors to appear on the cell surface.
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What is the current consensus on intra-Golgi trafficking? (2010)
1. Cisternal maturation explains how cargo moves through the Golgi.
2. The mechanism of enzyme retention is still debated — both COPI vesicle shuttling and direct diffusion have supporters.
