Secretory Pathway Flashcards
(31 cards)
1
Q
What is the Function of COPII Coat?
A
- Transporting proteins from the ER -> Golgi. this involves:
- Recognizing cargo at the ER membrane.
- Forming vesicles that bud off from the ER.
- Uncoating after vesicle formation, allowing the vesicles to fuse with the next compartment.
- vesicles fuse together to form a vesicular-tubular cluster (VTC).
- The VTC moves to the Golgi along MTs using dynein.
- Finally, the VTC fuses with the golgi membrane.
2
Q
What are the signals COPII receives for cargo selection?
A
- recognizes specific signals on the cytoplasmic parts of ER proteins.
- two phenylalanines at the C-terminus.
- cluster of acidic amino acids (must be on cytoplasmic side, but does not have to be at the end)
3
Q
What is the structure of COPII coat?
A
- Sar1 (small GTPase)
- key regulator of coat assembly and disassembly.
- in the cytoplasm in its inactive GDP-bound form.
- activated by Sec12 (a GEF), which replaces GDP with GTP.
- Sar-GTP inserts into the ER membrane and recruits the inner coat. - Inner COPII subunits: Sec23/24
- Sec23/24 binds to both Sar1 and cargo proteins.
- helps select and concentrate cargo for transport. - Outer COPII subunits: Sec13/21
- binds to Sec23/24.
- drives membrane bending and vesicle budding.
4
Q
What are the steps in COPII vesicle formation and uncoating?
A
- Sar1-GDP in the cytoplasm is activated by Sec12, which loads it with GTP.
- Sar1-GTP binds the ER membrane.
- Sar1-GTP recruits the inner coat (Sec23/24), which binds cargo.
- Outer coat (Sec13/31) binds to Sec23/24 and causes the membrane to curve and bud off.
- After budding, Sar1 hydrolyzes its GTP, becoming Sar1-GDP.
- Sar1-GDP detaches from the membrane, and the COPII coat is released (uncoating).
- The uncoated vesicle is then ready to fuse with the VTC.
5
Q
What is the trafficking process from ER -> Golgi?
A
- ER -> Vesicular-Tubular Cluster (VTC)
- VTC -> Golgi.
The transport uses COPII-coated vesicles, which carry proteins from ER to Golgi.
Step 1: ER to VTC
- COPII vesicle formation:
COPII vesicles bud from ER exit sites, these vesicles carry selected cargo to the next compartment
—> Role of Rab1 and p115:
- Rab1 (small GTPase) loaded onto COPII vesicles.
- Rab1 recuits p115, golgin tethering factor.
- p115 helps cluster COPII vesicles together by tethering them.
-The tethered vesicles fuse with each other using NSF and Snare proteins.
- fusion creates VTC, also called pre-Golgi intermediate.
Step 2: VTC to Golgi
- VTC transported along microtubules toward golgi.
- movement powered by Dynein (moves toward minus-end (cell center – where golgi located) and Dynactin (protein complex that works with dyenin to enhance transport)
- once at golgi, VTC fuses with golgi membrane, delivering its cargo into the golgi for further processing.
6
Q
What are the function of cargo receptors?
A
- Cargo receptors are transmembrane proteins that help soluble ER proteins (which cannot bind COPII directly) exit the ER by packaging them into COPII-coated vesicles.
- After delivering cargo to the Golgi, cargo receptors are recycled back to the ER using COPI-coated vesicles, maintaining the directionality and reuse of transport components.
7
Q
What characteristics to most cargo receptors have?
A
- A COPII-interaction signal, usually two phenylalanines (FF) at the C-terminus, allowing ER exit.
- A COPI-interaction motif, often a di-lysine sequence (-KKXX), which allows return from VTC or Golgi to the ER via retrograde transport.
- pH-sensitive cargo binding mechanism:
-> They bind cargo at the neutral pH of the ER lumen.
-> They release cargo in the slightly acidic pH of the Golgi or VTC lumen.
8
Q
What is an example of a cargo receptor?
A
ERGIC53 is a cargo receptor that binds N-linked oligosaccharides and has both COPII (FF) and COPI (KK) motifs in its -KKFF tail.
9
Q
What are forward (anterograde) cargo receptors?
A
- bind cargo in the ER (neutral pH) and release it in the Golgi or VTC (slightly acidic pH)
Usually contain:
- COPII-binding signals (commonly FF at the C-terminus) for ER exit.
- COPI-binding motifs (e.g., -KKXX) to be returned to the ER via retrograde transport.
10
Q
What are key examples of anterograde cargo receptors?
A
- ERGIC53
- Recognizes high-mannose N-linked oligosaccharides.
- Participates in anterograde (via COPII) and retrograde (via COPI) transport.
- Binds cargo at neutral pH (ER) and releases it at slightly acidic pH (VTC/Golgi). - p24 family:
- Binds GPI-anchored proteins in the ER and releases them in VTC/Golgi. - Surf4:
- Recognizes specific N-terminal tripeptides on some soluble secretory proteins.
11
Q
What is an example of a reverse (retrograde) cargo receptor?
A
- KDEL receptor
- Specialized to retrieve ER-resident lumenal proteins (which may escape to the Golgi by accident).
- Recognizes the KDEL sequence (Lys-Asp-Glu-Leu) on escaped proteins.
- Binds cargo at the acidic pH of the Golgi, then releases it in the neutral pH of the ER.
- Contains COPI-binding motifs, allowing it to travel back to the ER in COPI-coated vesicles.
12
Q
What is the role of COPI/Arf1 in Golgi to ER trafficking?
A
- Arf1 is a small GTPase that controls COPI recruitment to membranes.
- Arf1-GDP in the cytoplasm is activated by the GEF GBF1, which promotes exchange of GDP for GTP.
- Arf1-GTP inserts into the membrane and recruits COPI coat proteins.
- COPI then binds to sorting signals on cargo receptors and promotes membrane curvature and vesicle budding.
- ArfGAP proteins (e.g., ArfGAP1 or ArfGAP2) then stimulate GTP hydrolysis by Arf1.
- When Arf1 becomes GDP-bound, it dissociates from the membrane, causing COPI to uncoat, allowing the vesicle to fuse with the ER.
13
Q
How does differential centrifugation work?
A
- Used to separate cell components based on size and shape.
- Starts with a homogenized cell suspension.
- Particles are separated by increasing centrifugal force in sequential steps.
- Larger and heavier components (e.g., nuclei) sediment first; smaller ones (e.g., ribosomes) sediment later at higher speeds.
- Separation is based on sedimentation coefficient.
- Does not produce very pure fractions, because components of similar size may co-sediment.
14
Q
How does density gradient centrifugation work?
A
- Improved method for separating cellular components.
- Uses a density gradient (e.g., sucrose or metrizamide) in the centrifuge tube.
- Gradient is less dense at the top and more dense at the bottom.
- Particles move during centrifugation until they reach a point where their buoyant density equals the surrounding gradient (equilibrium point).
- Separation is based on density, not size or shape.
- Produces purer fractions of organelles.
- Gradients can be continuous or discontinuous.
- Fractions can be collected by perforating the bottom of the tube.
15
Q
What was the strategy for selecting Sec Mutants in Yeast? (Schekman’s Approach)
A
- Mutagenesis and Initial Growth:
- Yeast cells were exposed to a mutagen and grown at 24°C.
- It was hypothesized that cells unable to secrete proteins would accumulate more protein, increasing their density.
- Temperature Shift:
- Yeast were then shifted to 37°C for 3 hours.
- Cells unable to secrete proteins would accumulate more intracellular protein and become extra-heavy. - Density Gradient Centrifugation:
- The yeast cells were separated by density gradient centrifugation based on their weight, isolating the extra-heavy (mutant) yeast. - Reversal to Permissive Temperature:
- The cells were then shifted back to 24°C, assuming that continued blockage of secretion at high temperature would lead to yeast cell death. - Screening and Characterization:
- Colonies that grew at 24°C were screened and further characterized to identify Sec mutants.
- Mutations in 23 genes were identified in the first screen, which were later expanded.
16
Q
How was Electron Microscopy used to sort complementation groups?
A
- sec mutants divided into 23 complementation groups.
- mutations in different genes would complement each other when combination in a diploid cell, but mutations in the same gene would not.
- Haploid yeast containing different sec mutations were mated to form diploid yeast. If mutations were in different genes, the diploid progeny would be normal, even at the non-permissive temperature (37°C).
- If mutations were in the same gene, the diploid progeny would still show the temperature-sensitive phenotype.
THEN
- EM was used to further study the 23 complementation groups.
- The Sec mutants were sorted into categories based on their subcellular defects at the non-permissive temperature:
1. ER accumulation.
2. Golgi accumulation (showed enlarged golgi membranes - “Berkely bodies”)
3. Vesicle accumulation
17
Q
How was COPII coat initially identified?
A
- Schekman lab developed a system where vesicles could be formed in vitro from purified ER membranes.
- this process required: ER membranes, Cytosol, ATP, GTP, and other necessary factors.
- a key step in the discovery was the use of a non-hydrolyzable GTP analog, which prevented GTP hydrolysis. -> this allowed coated vesicles to accumulate, since vesicle uncoating is normally triggered by GTP hydrolysis.
- accumulated vesicles were isolated and analyzed.
- vesicles tested for presence of Sec proteins (which are involved in ER to Golgi trafficking) using immunogold labeling.
- This approach identified the components of the COPII coat and established their role in budding vesicles from the ER.
18
Q
What is the structure of the Golgi Apparatus and domains?
A
- golgi apparatus resembles a stack of pancakes (layered cisternae) and has distinct polarity.
- stacks arranged in long ribons with disorganized areas between stacks.
- it is divided into three main domains:
- cis-golgi: closest to ER; where cargo enters.
- medial-golgi; central region, where further modification of cargo occurs.
- trans-golgi: where cargo exits to its final destination (often called trans-Golgi network (TGN).
- As cargo proteins move from cis to trans, they are sequentially modified by different enzymes housed in specific golgi compartments.
- compartmentalized structure of the Golgi ensures that each modification step occurs in the correct order.
19
Q
Explain the function of ‘sequential modification of N-linked oligosaccharides’ in the golgi apparatus?
A
- N-glycans first added in ER.
- Further processes in the Golgi by a series of enzymes arranged in the order of their activity.
20
Q
How does O-linked glycosylation work in the Golgi apparatus?
A
- mostly in medial golgi
- begins with N-acetyl-galactosamine added to serine or threonine residues (often near glycine).
- can continue with addition of more sugars, especially proteoglycans, leading to long, repeating sugar chains.
- chains may be sulfated in the Golgi.
21
Q
What is the function of proteoglycan glycosylation in the Golgi?
A
- Essential for forming components of the extracellular matrix.
- Involves extensive sugar chain addition and modification.
22
Q
Explain how proteolytic modifications of proteins takes place in the golgi?
A
- Occurs mainly in the late Golgi or trans-Golgi network (TGN).
- The TGN is slightly acidic (pH ~6), which supports the function of acid proteases like furin.
- These proteases cleave and activate secreted or lysosomal proteins.
23
Q
How are secretory granules produced in the golgi?
A
- The acidic pH in the TGN helps in aggregating secreted proteins, such as digestive enzymes.
- These proteins are packed densely into granules for secretion.
- Further pH drop in granules enhances aggregation and volume reduction.
24
Q
How does the golgi sort proteins to various destinations?
A
- The trans-Golgi network plays a major role in directing proteins to their final destinations—lysosomes, plasma membrane, or secretion outside the cell.
25
What are the three main models for how cargo moves through the Golgi apparatus?
1. Direct connections model.
- suggest that the golgi cisternae are connected, allowing cargo to move by diffusion thtrough inter-cisternal tubules or channels.
2. Vesicular transport model.
- proposes that each cisterna is a separate compartment, and small vesicles carry cargo from one cisterna to the next.
- requires vesicle formation, budding, and fusion with the next cisterna.
3. Cisternal maturation model.
- cisternae themselves move forward from the cis to the trans face, carrying cargo with them.
- As they move, enzymes are recycled backward via vesicles to maintain compartment identity.
- Favored by electron microscopy, which showed trans cisternae peeling away—suggesting that cisternae mature and disassemble.
26
What evidence supported cisternal maturation model?
- Algal scales and procollagen are too large to fit into small vesicles. Yet they move through the Golgi, suggesting cisternae must carry them, as vesicles would be too small.
- Live-cell imaging shows cargoes present in a cisterna only briefly, consistent with the idea that cisternae move and mature.
- However, a challenge for this model is explaining how Golgi enzymes remain localized in different cisternae, since the enzymes must be actively recycled.
27
How does rothman's in vitro system work? what was the purpose?
- developed a cell-free system using Golgi membranes from two populations of CHO cells:
1. one expressed VSG-G protein but lacked the enzyme GlcNAc transferase.
2. the other had GlcNAc transferase, but no VSV-G.
When the two Golgi populations were mixed with cytosol, ATP, and radioactive GlcNAc, the VSV-G became modified - suggesting it had moved into the compartment with the enzyme.
- VSV-G was immunoprecipitated, and GlcNAc incorporation was detected using: SDS-PAGE followed by radioactivity measurement.
28
How was COPI coat identified?
- Using the in vitro system, blocking GTP hydrolysis with a non-hydrolyzable GTP analog (GTPγS) caused vesicles to become coated.
- Coat proteins purified and identified.
- A small GTPase called Arf was found to be necessary for coat formation.
29
What is Membrane Fractionation?
- It’s a multi-step purification process that usually involves centrifugation (spinning samples at high speeds) to separate different cellular components based on size and density.
- First developed in the 1960s–1970s to isolate organelles like the Golgi apparatus, ER, mitochondria, etc.
- The Rothman lab improved this method to obtain functional Golgi fractions—meaning that the purified Golgi could still support cargo transport in experimental conditions.
30
Why is Membrane Fractionation Important?
It’s essential for creating a cell-free (in vitro) system, which allows researchers to study specific cellular processes outside of living cells. For this, you need:
1. Purified membranes or organelles (e.g., Golgi via membrane fractionation).
2. Cytosol and energy molecules like ATP and GTP.
3. A cargo protein and an assay to detect if transport occurs.
31
How was membrane fraction applied to Rothman's work?
- Rothman used membrane fractionation to isolate two different populations of Golgi membranes.
- These Golgi membranes were then used in his in vitro assay to study intra-Golgi transport, leading to discoveries like:
1. How cargo moves through the Golgi.
2. The role of cytosolic factors and energy.
3. The discovery of COPI vesicle coats.
