Lecture 7 - Vesicular Trafficking II

Question 1

Docking and Fusion are Separate Processes. Give an example of where this is used in a biological system.

(2 Points)

Answer 1

Neurotransmitter Release:
* Complexin binds to partially assembled trans-SNARE complex, preventing membrane fusion until calcium signalling induces conformational change in synaptotagmin, allowing it to release inhibitory effect
* Complexin binding allows neurotransmitter release to be primed, but not occur until Ca2+ signalling occurs

Question 2

Give an Example of a SNARE-like fusion process in Viruses.

Answer 2

HIV:
* Following its Insertion into the host membrane, the HIV fusion protein collapses to form a trans-SNARE like complex (6 helices), which triggers fusion

Question 3

(i) Why do SNAREs need to be separated?
(ii) How does this occur?

Answer 3

(i) trans-SNARE complexes are very stable, therefore they must be separated by ATP Hydrolysis in order to be reused (important for regulating vesicle fusion)

(ii) Occurs via the action of the NSF complex

Question 4

What is the Structure of the NSF Complex

(2 Points)

Answer 4

• Dodecamer (12 Subunits) organised into two rings of six subunits (D1/D2)
• N-terminal domains of D1 interact with SNAP25 to carry out SNARE separation

Question 5

Describe the Mechanism of SNARE Separation, including:
(i) SNAP-SNARE Interaction
(ii) Energy requirement

(2 Points)

Answer 5

1. SNAP (Accessory Protein) interacts with the SNARE proteins via its conserved patches of +ve charge (interacts with -ve charge of SNAREs)
2. N-domains of NSF D1 subunits interact with SNAP proteins, then each subunit (AAA ATPase) sequentially hydrolyses ATP to provide energy to pull apart SNAREs

Question 6

(Why do Properties Change)

Compare the properties of Rab GTPases when bound to different nucleotides

(3 Points)

Answer 6

GDP-bound = soluble and inactive
GTP-bound = membrane-associated and active
* Due to conformational change that flips out amphipathic helix and exposes prenyl lipid group

Question 7

Summarise the mechanism of Rab targeting

(3 Points)

Answer 7

• Rab-GDP is converted to Rab-GTP by GEF in membrane of donor compartment, resulting in insertion into membrane
• Rab-GTP is trafficked in vesicle membrane to target compartment, where it interacts with Rab effector (e.g., EEA1)
• RabGTP-effector interaction brings vesicle into close enough proximity to allow trans-SNARE complex to form

Question 8

How is Specificity of RabGTP-effector interaction established?

Answer 8

Rab effector proteins (e.g., EEA1, Rebenosyn-1) contain localisation domains

Question 9

What is the function of the GDP dissociation inhibitor?

Answer 9

Prevents dissociation of GDP from RabGDP in absence of the GEF, thereby preventing random insertion into compartment membranes

Question 10

Define the FYVE domain in terms of:
(i) Structure
(ii) Localisation
(iii) Function

Answer 10

(i) Coordinates Zn2+ ions, which stabilise the domain fold
(ii) Localises to the Late Endosome due to its enrichment in PI-3P headgroups
(iii) Interaction with PI-3P brings domain into close enough proximity to membrane for insertion of hydrophobic residues (e.g., Lys)

Question 11

What other roles can RabGTPases play in the cell?

Answer 11

• Rab proteins can be involved in recruitment of effector proteins to different cellular compartments, which is particularly important during endosomal maturation (e.g. can recruit GEFs specific for other Rab proteins)

Question 12

How does the cell ensure multi-subunit proteins are correctly assembled before transport out of ER?

Answer 12

Multi-subunit proteins (e.g., IgG) may contain retention markers, which will be bound by chaperones to prevent transport until the other subunits bind and displace them

Question 13

Membrane Inserted vs Soluble

Compare how different ER-resident proteins are retrieved from Golgi

(2 Points)

Answer 13

• Membrane Inserted (e.g., KDEL receptor) - contain the KKXX motif, which interacts with COPI to induce vesicle budding
• Soluble (e.g., BIP) - contain KDEL motif, which is recognised by KDEL receptor and leads to recruitment into COPI vesicle

Question 14

How is the affinity of KDEL receptors regulated to allow return of ER-resident soluble proteins?

(2 Points)

Answer 14

• In Golgi - high affinity due to low pH
• In ER - low affinity due to high pH

Question 15

What other protein retrieval mechanisms exist in cells?

(2 Points)

Answer 15

• Size Exclusion - ER proteins may bind together, becoming too large for incorporation into COPII coated vesicles (e.g., Kin Recognition)
• Resident Golgi/ER membrane proteins - have shorter TM segments than PM proteins, hence cannot be inserted into PM

Question 16

Describe how Mannose-6-Phosphate (M6P) directs transport of Lysosomal Hydrolases

Answer 16

• M6P - typically signals cargo for transport into the lysosomes via interactions with M6PR
• Signal for M6P addition - present in primary sequence of lysosomal hydrolases
• Motif-Receptor Interactions - low affinity, hence multiple M6Ps may be required for high affinity interactions

Question 17

How do M6PRs recruit cargo into budding vesicles?

Answer 17

Cytoplasmic tails contain multiple binding sites for adaptor proteins (e.g., APs), leading to recruitment of clathrin coat