IV. Cell Biology | 61. The protein secretory pathway; role of the rab cycle in the regulation of vesicular transport

Question 1

I. Basics
1. Proteins are transported and secreted via ___

Answer 1

vesicles

Question 2

I. Basics
2. Every cell is bound by ____

Answer 2

a membrane (PM)
(that separates the cytosol from the extracellular matrix)

Question 3

I. Basics
3. A eukaryotic cell is also separated by membranes into ____

Answer 3

subcellular compartments = organelles

Question 4

I. Basics
4. How is communication between intracellular compartments and extracellular matrix mantained?

Answer 4

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

Question 5

II. Vesicular transport
1. What is Vesicular transport?

Answer 5

The transport between membrane-enclosed compartments (green arrows in the picture above) – between extracellular and intracellular environment

Question 6

II. Vesicular transport
2. What does Vesicular transport need?

Answer 6

Always need a donor membrane (+ target membrane where the fusion occurs)

Question 7

II. Vesicular transport
3. What is the role of Vesicular transport?

Answer 7

• Necessary to maintain the function of different compartments
  (ex: lysosome requires hydrolytic enzymes.
• If they are not delivered, the lysosome cannot execute its function)

Question 8

II. Vesicular transport
4. Why is the specificity of vesicular transport very important?

Answer 8

1. Needs to be specific for what and where it transports
2. Reason for specificity is the character of a compartment
   which is primarily defined by the composition of the membrane enclosed space
3. Membrane is aided by markers that guide incoming traffic – make sure
   vesicles fuse only with the correct compartment

Question 9

II. Vesicular transport
5. What are 4 Steps of vesicle formation?

Answer 9

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

Question 10

II. Vesicular transport
6. What are the 3 types of protein coats?

Answer 10

3 different types of protein coats: clathrin, COP-I and COP-II

Question 11

II. Vesicular transport
7. Three different types of protein coats: clathrin, COP-I and COP-II
=> Give a general description about their role

Answer 11

1. Clathrin-coated vesicles mediate transport from Golgi -> lysosome (anterograde) + from PM -> endosome (retrograde)
2. COP-I coated vesicles mediate transport from Golgi -> ER (retrograde)
3. COP-II coated vesicles mediate transport from ER -> Golgi (anterograde)

Question 12

II. Vesicular transport
8. What are the 2 layers of protein coat?

Answer 12

1. Inner coat layer: selects the appropriate cargo and allows coat proteins to assemble on it
2. Outer coat layer: assembles like a curve (basketlike lattice) that deforms the membrane path and shapes the vesicle

Question 13

III. Clathrin-coated vesicles
1. What is the structure of Clathrin-coated vesicles?

Answer 13

• 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

Question 14

III. Clathrin-coated vesicles
2. How do Clathrin-coated vesicles grow?

Answer 14

• 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

Question 15

III. Clathrin-coated vesicles
3. What is the role of adaptin?

Answer 15

recognizes the cargo receptor by phosphatidylinositol lipids – PIPs

Question 16

III. Clathrin-coated vesicles
4. What are the 4 types of adaptin?

Answer 16

• AP1: located in trans-Golgi, carries lysosomal hydrolase
• AP2: located in PM, functions during endocytosis
• AP3: transport to lysosomes
• AP4: less known

Question 17

III. Clathrin-coated vesicles
5. What is Dynamin?

Answer 17

Dynamin (and associated proteins) is a cytosolic protein that catalyzes the budding off of the vesicle from the membrane

Question 18

III. Clathrin-coated vesicles
6. What is the structure of Dynamin?

Answer 18

Dynamin contains a
phosphatidylinositol binding domain which tethers the protein to the membrane

Question 19

III. Clathrin-coated vesicles
7. What is the mechanism of dynamin?

Answer 19

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

Question 20

III. Clathrin-coated vesicles
8. How is Uncoating of the clathrin vesicle catalyzed?

Answer 20

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

Question 21

IV. COP-I coated vesicles
1. What is the role of COP-I coated vesicles?

Answer 21

Transports proteins from trans-Golgi to cis-Golgi + the ER
=> a retrograde pathway is used if the proteins are folded wrong

Question 22

IV. COP-I coated vesicles
2. What is the ARF1?

Answer 22

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)

Question 23

IV. COP-I coated vesicles
3. How are COP-I coated vesicles formed?

Answer 23

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

Question 24

IV. COP-I coated vesicles
4. What is the role of Sec7?

Answer 24

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

Question 25

IV. COP-I coated vesicles 5. What happen Once AFR1-GTP is active?

Answer 25

Once AFR1-GTP is active, it releases p23 and binds the Golgi membrane directly

Question 26

IV. COP-I coated vesicles 6. What is coatomer? How is it formed?

Answer 26

p23 then binds p24 -> form a heterosexual-oligomer where the costumer (COP-1) can assemble +) coatomer = protein complex that coats membrane-bound transport vesicles

Question 27

IV. COP-I coated vesicles 5. What is the role of BAR-domain proteins and DAG?

Answer 27

When the vesicle is big enough, BAR-domain proteins and DAG will be responsible for the budding off of the vesicle

Question 28

IV. COP-I coated vesicles 6. For disassembly of the coat, what need to occur?

Answer 28

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

Question 29

V. COP-II coated vesicles 1. What is the role of COP-II coated vesicles?

Answer 29

Transport proteins from the ER to the Golgi (cis) = anterograde pathway

Question 30

V. COP-II coated vesicles 2. How is COP-II coated vesicles formed?

Answer 30

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

Question 31

V. COP-II coated vesicles 2. What is the role of Sar1?

Answer 31

Sar1 (Ras-like G-protein) similar to ARF, is inactive when bound to GDP

Question 32

V. COP-II coated vesicles 3. How is Sar1 activated?

Answer 32

Sec 12 (= Sar1-GEF) catalyzes GDP to GTP -> activating Sar1-GTP

Question 33

V. COP-II coated vesicles 5. What is the role of Sar1-GTP?

Answer 33

- 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

Question 34

V. COP-II coated vesicles 6. What do Sec23 and Sec24 do?

Answer 34

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

Question 35

V. COP-II coated vesicles 7. What do Coat proteins Sec13/31?

Answer 35

Coat proteins Sec13/31 will bind to the adaptor complex Sec24/23 to form the outer coat layer of the COP-II vesicle

Question 36

VI. Rab cycle 1. Give a summary of Rab cycle?

Answer 36

- 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

Question 37

VI. Rab cycle 2. What is the role of Rab?

Answer 37

- 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

Question 38

VI. Rab cycle 3. What are the 5 steps of Rab cycle?

Answer 38

Question 39

VI. Rab cycle 3. What is the role of GDI (GDP dissociation inhibitor)?

Answer 39

- 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

Question 40

VII. Tethering of vesicle to acceptor/target membrane 1. What are the 3 steps of Tethering of vesicle to acceptor/target membrane?

Answer 40

1. Recognition 2. Docking 3. Fusion

Question 41

VII. Tethering of vesicle to acceptor/target membrane 2. What happen in step 1: Recognition of Tethering of vesicle to acceptor/target membrane?

Answer 41

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

Question 42

VII. Tethering of vesicle to acceptor/target membrane 3A. What happen in step 2: Docking of Tethering of vesicle to acceptor/target membrane?

Answer 42

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

Question 43

VII. Tethering of vesicle to acceptor/target membrane 3A. What happen in step 2: Fusion of Tethering of vesicle to acceptor/target membrane?

Answer 43

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

Question 44

VII. Tethering of vesicle to acceptor/target membrane 4. What is the role of SNAREs?

Answer 44

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