Vesicle Trafficking

Question 1

How are vesicles formed

Answer 1

1. Coat proteins assembly at membrane forces bilayer to begin to bend
2. As they gather at the membrane, coat proteins may also select cargo to be packaged into the vesicle
3. More coat protein binding results in the formation of the sphere of membrane
4. Once coated, vesicle pinched off. Coat is then detached and cargo-filled vesicle is transported to destination

This process helps to concentrate proteins into a special patch on the membrane for designated transport

Question 2

What are the 3 types of archetypal vesicle coat

Answer 2

Clathrin, COPI and COPII drive the formation of transport vesicles by polymerising on cellular membranes
COPII covers vesicles emanating from the ER
COPI surrounds vesicles originating from Golgi
Clathrin surrounds those from the plasma membrane

(Coats around vesicles)

Question 3

What is a vesicle

Answer 3

Small lipid membrane sack that carried cargo between intracellular compartments

Question 4

What is the ERGIC

Answer 4

Sits in between ER and Golgi

Question 5

What does COPIdo

Answer 5

Transport from Golgi to ER

Question 6

What does COPII do

Answer 6

Transport from the ER to the Golgi

Question 7

What are the core principles of vesicular trafficking

Answer 7

Cargo selection-> specific molecules inside the cell are identified for transport
Cargo receptor binding
Coat proteins ecruitment
Membrane budding
Unloading
Vesicle transport
Fusion with target compartnment

Question 8

How is cargo transport from the ER to the Golgi via COPII

Answer 8

1. Sec12p is a GEF that exchanges GDP to GTP in Sar1p
2. Sar1p is activated by GTP (inserts helix to embed in ER membrane)
3. Activated Sar1p acquires Sec23/24p to form core inner coat of COPII coat
4. Acquires outer coat scaffold complex Sec13/31p
5. Budding/pinching off (vesicle pinches off)
6. GTP hydrolysis by Sar1p releases coat and vesicle

Question 9

How is cargo transported back to the ER from the Golgi via COPI

Answer 9

• COPI coat formed from 7 polypeptides
• Arf1 GTPase similar to Sar1 from COPII process
• Guanine exchange factors involved
  (Process isn’t as well understood as COPII

Question 10

How does transport from the outside of the cell to inside the cell via clathrin coated vesicles from the plasma membrane

Answer 10

• more complex that COPI and COPII
• Arf GTPase also involved to recruit adaptors
• clathrin layered onto coating to crease clathrin-coated vesicles composed of triskelions
• each triskelion composed of three clathrin heavy chains interacting at their C-termini
• each heavy chain has a 25kDa light chain tightly bound
• heavy chains provide the structural backbone of the clathrin lattice and the light chains thought to regulate formation and disassembly of lattice

Question 11

What are the steps for the formation of a clathrin coated vesicle

Answer 11

1. Arf GTPase initiates assembly recruiting co-factors
2. Adaptor proteins give specificity
3. Dynamin pinches off vesicle
4. Vesicle unloading mediated by Hsc70 and auxiliary
5. Vesicle moves to target

Question 12

What is Dynamin and what is its role (how is it involved in clathrin-coated vesicle formation)

Answer 12

-Dimeric GTPAse recruited to membrane
- GTP hydrolysis leads to a power strike and increased constriction
- fission of membrane where the membrane stress is the largest
- disassembly of the oligomer and recycling of Dynamin

Question 13

When is Dynamin not functional

Answer 13

Not functional at high temperatures
Therefore it can’t be used to pinch off vesicles
This could lead to paralysis as there’s no vesicle regeneration at synapses

Question 14

What is the structure of clathrin

Answer 14

36 triskelions make up a hexagonal clathrin cage
Triskelions are three legged heteropolymers (composed of 3 heavy and 3 light chains)

Question 15

What pathways can clathrin coated vesicles from the PM take

Answer 15

Can take multiple pathways around the cell

Question 16

What would happen if you block vesicle fusion

Answer 16

Paralysis (mentioned in another flash card)

Question 17

What theories are there for the vesicle fusion process

Answer 17

The SNARE hypothesis

Question 18

What is the SNARE hypothesis

Answer 18

Series of molecules discovered to play a role in vesicle fusion (NSF, alpha SNAP, and alpha SNAP receptor or SNARES)
SNARE hypothesis= each type of transport vesicle carries a specific v-SNARE that binds to a cognate t-SNARE on the target membrane
Binding between v and t-SNARES makes a stable four helix bundle
1 helix contributed by the v-SNARE the other 3 are contributed by the oligomeric t-SNARE

Question 19

What do SNAREs do

Answer 19

Promote fusion by overcoming major energy barrier to fusion
Help ensure specificity of membrane fusion
Different/t SNARE complexes form at different steps of intracellular transport

Question 20

What proof is there of SNARE specificity

Answer 20

In vitro liposomes with SNARE partners
SNAREs alone can fuse with vesicles
Highly specific interaction of v and t SNARES

Question 21

What is the role of Rab GTPases

Answer 21

Guide vesicle targetting- lots of different forms each specific for different cargos
Active Rab-GTP binds Rab effectors for movement for tethering vesicles to membranes
Includes motor proteins or tethering proteins or SNAREs-coupling tethering to fusion

Question 22

How can some neurotoxins work

Answer 22

They work by inhibiting SNARE complex formation
E.g tetanus, botulinum toxins hydrolyse peptide bonds with SNARE

Question 23

What is the extracellular matrix (ECM)

Answer 23

Cells in tissues are
organised into complex structures surrounded by the ECM
Fundamental role in development
is a structural support network made up of many proteins and macromolecules

Question 24

What is the basal lamina and its role

Answer 24

Thin mesh of ECM molecules important in tissue repair an ddevelopment
Contains multi-adhesive matrix proteins e.g lamina
Type IV collagen (principle structural component)
Perelecan (proteoglycan), bind diverse partners, cross link components

Question 25

What is connective tissue and what is its role *

Answer 25

Is the abundant environment that cells in tissues live in
Varied in form e.g tough and flexible = tendon, hard and dense=bone, shock absorbing = cartilage

Question 26

What are some examples of secreted proteins including those involved in cell-cell adhesion and the ECM*

Answer 26

laminin
fibronectin
collagens

Question 27

what are cell-cell junctions

Answer 27

they link cells to each other in tissues