Lecture 6 - Vesicular Trafficking I

Question 1

Why do cells need to perform vesicular transport?

(4 Points)

Answer 1

• Nutrients
• Communication with environment (i.e. Receptor-mediated Endocytosis)
• Regulation of Protein Delivery
• Coordination of Protein Synthesis, Modification and Delivery

Question 2

(i) Define Anterograde/Retrograde Transport

(ii) Why must they be balanced?

(2 Points)

Answer 2

(i):
* Anterograde = Towards cell periphery/outside
* Retrograde = Towards cell centre

(ii) to maintain membrane levels at different compartments

Question 3

Define the Three types of coated vesicles, including the type(s) of trafficking they are involved in

Answer 3

1. Clathrin - receptor-mediated endocytosis, but also Golgi to endosome transport
2. COPI - typically involved in retrieval pathways, but also involved in budding from the golgi
3. COPII - only present on vesicles budding from ER

Question 4

Describe:
(i) Clathrin Structure
(ii) Clathrin-coated Vesicle Formation
(iii) Adaptins

Answer 4

(i) Triskelion structure composed of 3 Heavy chains (180kDa) and 3 light chains (~40kDa)
(ii) Clathrin monomers self-polymerise to produce a “basket-like” structure, whose curvature deforms the membrane
(iii) Heterotetrameric (a,B,y,µ) Cargo Adaptors, which recruit cargo into clathrin-coated vesicles

Question 5

Compare (i) COPI and (ii) COPII coat proteins

(4 Points)

Answer 5

(i) COPI-coated vesicles are formed from the COPI coatamer complex (multiple different subunits, with several sharing homology with APs)

(ii) COPII-coated vesicles are formed from two sub-complexes:
* Sec23/24 tetramer (cargo recruitment, no homology to APs)
* Sec13/31 complex (self-polymerises into a “basket-like” structure)

Question 6

How do Adaptins recruit cargo into Clathrin-coated vesicles?

Answer 6

Recognise cargo via cargo receptors, and has binding sites for Clathrin recruitment

Question 7

How is Coat Assembly regulated to ensure balanced vesicle trafficking? How does this work?

(3 Points)

Answer 7

• Controlled by monomeric coat-recruitment GTPases (e.g., ARF, Sar1), which are present in high concentrations in an inactive GDP-bound state
• Sar1-GDP interacts with a membrane-bound GEF (e.g., Sec12), causing GDP->GTP exchange that induces a conformational change in protein, exposing an amphipathic helix that inserts into membrane
• Sar1-GTP recruits adaptor proteins (e.g., Sec23/24), which recruit coat proteins

Question 8

What is Dynamin? How does it Function?

Answer 8

• Monomeric GTPase which is recruited to the neck of a budding vesicle (due to curvature), where it polymerises into RH helix
• Monomers in RH helix undergo simultaneous GTP hydrolysis, constricting neck to allow lipid exchange that severs budding vesicle

Question 9

How is Vesicular Trafficking Controlled to ensure fusion with correct compartment (Give two Types)?

Answer 9

• Vesicles carry surface proteins which interact with complementary surface proteins on the target compartment
• E.g., Soluble NSF Attachment receptors (SNAREs), Rabs

Question 10

1. What are SNAREs?
2. How do they facilitate highly specific membrane docking and fusion?

Answer 10

1. SNAREs - proteins with characteristic helical domains, that can be divided into v-SNAREs and t-SNAREs
   * v-SNARE/t-SNARE pairing - highly specific, with only certain combinations being allowed
2. v-SNAREs and t-SNAREs interact/intertwine to form a four helical bundle known as the trans-SNARE complex (very stable)
   * trans-SNARE complex brings membranes close enough together to allow lipid exchange and subsequent fusion