IV. Cell Biology | 61. The protein secretory pathway; role of the rab cycle in the regulation of vesicular transport Flashcards
I. Basics
1. Proteins are transported and secreted via ___
vesicles
I. Basics
2. Every cell is bound by ____
a membrane (PM)
(that separates the cytosol from the extracellular matrix)
I. Basics
3. A eukaryotic cell is also separated by membranes into ____
subcellular compartments = organelles
I. Basics
4. How is communication between intracellular compartments and extracellular matrix mantained?
Communication between intracellular compartments and the extracellular matrix must be maintained => achieved by transport mechanisms:
- Diffusion (gas molecules, small non-polar)
- Protein mediated transmembrane transport (by TM channels/receptors)
- Vesicular transport
II. Vesicular transport
1. What is Vesicular transport?
The transport between membrane-enclosed compartments (green arrows in the picture above) – between extracellular and intracellular environment
II. Vesicular transport
2. What does Vesicular transport need?
Always need a donor membrane (+ target membrane where the fusion occurs)
II. Vesicular transport
3. What is the role of Vesicular transport?
- Necessary to maintain the function of different compartments
(ex: lysosome requires hydrolytic enzymes. - If they are not delivered, the lysosome cannot execute its function)
II. Vesicular transport
4. Why is the specificity of vesicular transport very important?
- Needs to be specific for what and where it transports
- Reason for specificity is the character of a compartment
which is primarily defined by the composition of the membrane enclosed space - Membrane is aided by markers that guide incoming traffic – make sure
vesicles fuse only with the correct compartment
II. Vesicular transport
5. What are 4 Steps of vesicle formation?
1) When coat proteins (Clathrin, COP-I, COP-II) assemble at the membrane, they force the lipid bilayer to begin to bend
2) As they gather at the membrane, coat proteins may also select the cargo that is packaged into the forming vesicles
3) As more coat proteins are added, they shape the surrounding membrane into a sphere
4) Once a coated vesicle pinches off
-> the coat falls off, and the cargo-filled vesicle is ready to travel to its destination
II. Vesicular transport
6. What are the 3 types of protein coats?
3 different types of protein coats: clathrin, COP-I and COP-II
II. Vesicular transport
7. Three different types of protein coats: clathrin, COP-I and COP-II
=> Give a general description about their role
- Clathrin-coated vesicles mediate transport from Golgi -> lysosome (anterograde) + from PM -> endosome (retrograde)
- COP-I coated vesicles mediate transport from Golgi -> ER (retrograde)
- COP-II coated vesicles mediate transport from ER -> Golgi (anterograde)
II. Vesicular transport
8. What are the 2 layers of protein coat?
- Inner coat layer: selects the appropriate cargo and allows coat proteins to assemble on it
- Outer coat layer: assembles like a curve (basketlike lattice) that deforms the membrane path and shapes the vesicle
III. Clathrin-coated vesicles
1. What is the structure of Clathrin-coated vesicles?
- One clathrin molecule has 3 heavy chains and 3 light chains, forming a
three legged structure called triskelion - One leg of one clathrin molecule binds to the leg of another clathrin -> this is how they will cover the whole surface of the vesicle
III. Clathrin-coated vesicles
2. How do Clathrin-coated vesicles grow?
- Cargo molecules bind to cargo receptors (specific) in the cell membrane of the growing bud
- Clathrin molecules bind the cargo receptors via an adaptor protein complex called adaptin (recognizes the cargo receptor by phosphatidylinositol lipids – PIPs)
- Cargo receptor + adaptin complex = inner coat layer
III. Clathrin-coated vesicles
3. What is the role of adaptin?
recognizes the cargo receptor by phosphatidylinositol lipids – PIPs
III. Clathrin-coated vesicles
4. What are the 4 types of adaptin?
- AP1: located in trans-Golgi, carries lysosomal hydrolase
- AP2: located in PM, functions during endocytosis
- AP3: transport to lysosomes
- AP4: less known
III. Clathrin-coated vesicles
5. What is Dynamin?
Dynamin (and associated proteins) is a cytosolic protein that catalyzes the budding off of the vesicle from the membrane
III. Clathrin-coated vesicles
6. What is the structure of Dynamin?
Dynamin contains a
phosphatidylinositol binding domain which tethers the protein to the membrane
III. Clathrin-coated vesicles
7. What is the mechanism of dynamin?
Dynamin (GTPase activity) polymerize around the neck of the vesicle -> hydrolyzes GTP to GDP -> utilizes this energy to perform the release of the vesicle
- It brings the two ‘’leaflets’’ of the membrane close together, eventually allowing them to fuse and separate the vesicle from the donor membrane
- Uncoating of the clathrin vesicle is catalyzed by Hsc70-ATP auxillin, which hydrolyzes ATP and uses this energy to peel off the clathrin coat from the vesicle -> important that the uncoating does not occur before the vesicle has been formed
III. Clathrin-coated vesicles
8. How is Uncoating of the clathrin vesicle catalyzed?
Uncoating of the clathrin vesicle is catalyzed by Hsc70-ATP auxillin, which hydrolyzes ATP and uses this energy to peel off the clathrin coat from the vesicle
-> important that the uncoating does not occur before the vesicle has been formed
IV. COP-I coated vesicles
1. What is the role of COP-I coated vesicles?
Transports proteins from trans-Golgi to cis-Golgi + the ER
=> a retrograde pathway is used if the proteins are folded wrong
IV. COP-I coated vesicles
2. What is the ARF1?
ARF1 is a coat recruitment GTPase (Ras-like G-protein) that is responsible for assembly of the COP-1 coat on the Golgi membrane (cis-Golgi)
IV. COP-I coated vesicles
3. How are COP-I coated vesicles formed?
1) In the Golgi membrane, the ARF-GDP binds p23 homo-oligomers (monomers are the same)
2) Once it is bound, a GEF (guanine nucleotide exchange factor) called Sec7 exchanges the GDP with a GTP = activation of ARF1
3) Once AFR1-GTP is active, it releases p23 and binds the Golgi membrane directly
4) p23 then binds p24 -> form a heterosexual-oligomer where the costumer (COP-1) can assemble
+) coatomer = protein complex that coats membrane-bound transport vesicles
5) When the vesicle is big enough, BAR-domain proteins and DAG will be responsible for the budding off of the vesicle
6) For disassembly of the coat, the GTP on ARF1 is hydrolyzed to GDP by GAP (GTPase activating protein)
-> this ARF-GDP is inactive and can no longer bind the coatomer = disassembly
-> fusion of vesicle with target membrane
IV. COP-I coated vesicles
4. What is the role of Sec7?
Once the ARF-GDP binds p23 homo-oligomers (monomers are the same), a GEF (guanine nucleotide exchange factor) called Sec7 exchanges the GDP with a GTP = activation of ARF1
IV. COP-I coated vesicles
5. What happen Once AFR1-GTP is active?
Once AFR1-GTP is active, it releases p23 and binds the Golgi membrane directly
IV. COP-I coated vesicles
6. What is coatomer? How is it formed?
p23 then binds p24 -> form a heterosexual-oligomer where the costumer (COP-1) can assemble
+) coatomer = protein complex that coats membrane-bound transport vesicles
IV. COP-I coated vesicles
5. What is the role of BAR-domain proteins and DAG?
When the vesicle is big enough, BAR-domain proteins and DAG will be responsible for the budding off of the vesicle
IV. COP-I coated vesicles
6. For disassembly of the coat, what need to occur?
For disassembly of the coat, the GTP on ARF1 is hydrolyzed to GDP by GAP (GTPase activating protein)
=> this ARF-GDP is inactive and can no longer bind the coatomer = disassembly
=> fusion of vesicle with target membrane
V. COP-II coated vesicles
1. What is the role of COP-II coated vesicles?
Transport proteins from the ER to the Golgi (cis) = anterograde pathway
V. COP-II coated vesicles
2. How is COP-II coated vesicles formed?
1) Sar1 (Ras-like G-protein) similar to ARF, is inactive when bound to GDP
2) Sec 12 (= Sar1-GEF) catalyzes GDP to GTP -> activating Sar1-GTP
3) Sar1-GTP then binds to the ER membrane directly via its amphipathic helix (this helix is only available after Sar1 is bound to GTP)
4) Sar1 then recruits the adaptor complex Sec24-Sec23, forming the inner coat layer
5) Sec23 binds Sar1 (Sec23 will in the future function as a GAP) (GTPase activating protein) = hydrolysis of GTP to GDP)
6) Sec24 binds the cargo receptor in the ER membrane
7) Coat proteins Sec 13/31 will bind to the adaptor complex Sec24/23 to form the outer coat layer of the COP-II vesicle
V. COP-II coated vesicles
2. What is the role of Sar1?
Sar1 (Ras-like G-protein) similar to ARF, is inactive when bound to GDP
V. COP-II coated vesicles
3. How is Sar1 activated?
Sec 12 (= Sar1-GEF) catalyzes GDP to GTP -> activating Sar1-GTP
V. COP-II coated vesicles
5. What is the role of Sar1-GTP?
- Sar1-GTP then binds to the ER membrane directly via its amphipathic helix (this helix
is only available after Sar1 is bound to GTP) - Sar1 then recruits the adaptor complex Sec24-Sec23, forming the inner coat layer
V. COP-II coated vesicles
6. What do Sec23 and Sec24 do?
5) Sec23 binds Sar1 (Sec23 will in the future function as a GAP) (GTPase activating protein) = hydrolysis of GTP to GDP)
6) Sec24 binds the cargo receptor in the ER membrane
V. COP-II coated vesicles
7. What do Coat proteins Sec13/31?
Coat proteins Sec13/31 will bind to the adaptor complex Sec24/23 to form the outer coat layer of the COP-II vesicle
VI. Rab cycle
1. Give a summary of Rab cycle?
- The vesicles are bound to the motor protein on the cytoskeleton either directly or via adaptor proteins.
- Rab is an adaptor protein, also a GTPase (small monomeric G-protein) -> inactive when bound to GDP, active when bound to GTP.
- When Rab is bound to GTP it can bind the vesicle and mediate transport.
- Rab will bind to the vesicle while it is being formed, so it is located under the coat and will not dissociate when the coat disassembles
VI. Rab cycle
2. What is the role of Rab?
- Rab is an adaptor protein, also a GTPase (small monomeric G-protein) -> inactive when bound to GDP, active when bound to GTP.
- When Rab is bound to GTP it can bind the vesicle and mediate transport.
- Rab will bind to the vesicle while it is being formed, so it is located under the coat and will not dissociate when the coat disassembles
VI. Rab cycle
3. What are the 5 steps of Rab cycle?
VI. Rab cycle
3. What is the role of GDI (GDP dissociation inhibitor)?
- Rab is an adaptor protein, also a GTPase (small monomeric G-protein) -> inactive when bound to GDP, active when bound to GTP.
- When Rab is bound to GTP it can bind the vesicle and mediate transport.
- Rab will bind to the vesicle while it is being formed, so it is located under the coat and will not dissociate when the coat disassembles
VII. Tethering of vesicle to acceptor/target membrane
1. What are the 3 steps of Tethering of vesicle to acceptor/target membrane?
- Recognition
- Docking
- Fusion
VII. Tethering of vesicle to acceptor/target membrane
2. What happen in step 1: Recognition of Tethering of vesicle to acceptor/target membrane?
Recognition: the Rab protein in the vesicle binds the tethering protein = effector of Rab, in the target membrane.
- Different types of Rab proteins bind different types of tethering proteins -> increasing the specificity of the vesicle
VII. Tethering of vesicle to acceptor/target membrane
3A. What happen in step 2: Docking of Tethering of vesicle to acceptor/target membrane?
Docking: tethering proteins pull vesicles closer to the target membrane, so that the SNAREs can interact
- SNAREs attract SNAPs and NSF
- SNAP: stabilize the SNARE-complex
- NSF: is an ATPase that hydrolyzes ATP to ADP -> energy used to displace H2O from the interface between the vesicle and membrane
- SNARE complex forms spontaneously -> also releases energy used in H2O displacement
VII. Tethering of vesicle to acceptor/target membrane
3A. What happen in step 2: Fusion of Tethering of vesicle to acceptor/target membrane?
The tight SNARE-pairing forces lipid bilayers to come close and contact each other
-> fusion of the lipid layers
-> uptake of the vesicle + cargo is released into the IC space of the organelle
VII. Tethering of vesicle to acceptor/target membrane
4. What is the role of SNAREs?
SNAREs attract SNAPs and NSF
- SNAP: stabilize the SNARE-complex
- NSF: is an ATPase that hydrolyzes ATP to ADP -> energy used to displace H2O from the interface between the vesicle and membrane
