SNARE Regulatory Proteins

Question 1

SM proteins - what is their function, where were they first identified and what does ‘SM’ stand for?

Which specific SNARE do they interact with and in which configuration?

Answer 1

‘Secretory Mutant’ proteins - first identified in Yeast Screens.
These proteins interact with SNAREs and are essential for membrane fusion.

Question 2

Main SNARE regulatory protein (and yeast homologue) - name, where is it found and where does it mainly interact/ bind to exert its action?

Answer 2

Munc18 (human) and SEC1 (yeast) - first identified as binding partner of ‘closed’ conformation of syntaxin therefore thought to be negative regulator of SNARE mediated fusion

Question 3

What can be seen regarding vesicle docking in Munc18/syntaxin deletion? (2 separate experiments)

Answer 3

• Considerably slower fusion in the absence of Munc18 (as shown by measuring flourescence, where the concentration of flourescent molecules drop when vesicles fuse therefore so does the FRET signal)
• in Munc18 knockout, number of vesicles is the same but the number of docked vesicles is much much lower

> > > docking and fusion impacted!

Question 4

What was shown re Munc18 ‘clustering’ when Munc18 paired with GFP and what did this suggest?

Answer 4

Clusters of Munc18 are seen and many vesicles are located on top of these Munc18 clusters (or MICRODOMAINS)

> > > is Munc18 creating a DOCKING SITE for vesicles?

Question 5

Co-localisation of Munc18 and vesicles – what happens regarding this when Munc18 is knocked out?

Answer 5

•Co-localization studies show that vesicles ‘dock’ to munc-18 microdomains
>Subsequent studies showed that overexpression of Snap-25 in Munc18 rescues the ‘docking defect’

Question 6

SNAP25 ‘rescue’ effect - what was shown – what did this allow to conclude about the function of Munc18

Answer 6

-By overexpressing SNAP25 in Munc18 mutants we are able to RESCUE THE DOCKING DEFECT

-BUT SNAP25 only rescues DOCKING not FUSION in
munc18-1 null chromaffin cells

Question 7

What is meant by Munc18 NEGATIVELY regulates fusion?

Answer 7

Munc18-1 binding to ‘closed’ syntaxin by a specific binding site regulates ability of SNAREs to form complexes and consequently vesicles to dock to the plasma membrane and undergo fusion

Question 8

Munc13-1 is essential for fusion competence of

glutamatergic synaptic vesicles – how did data show this?

Answer 8

• Munc13(-/-) mice histological brain samples were taken.
• These could be stimulated with an electrode in neuron causing A.Ps to be fired – can record the glutamate-mediated currents (therefore secretion of NTs)
• Huge reduction in glutamate currents compared to WT and a reduction in the pool of readily releasable vesicles

Question 9

Munc13 cannot rescue Munc18 docking defect but is involved in fusion – what does this suggest?

Answer 9

Acts AFTER DOCKING but BEFORE FUSION —– INVOLVED IN A PRIMING REACTION

Question 10

The priming reaction – why is it important? What does it involve?

Answer 10

The priming step is RATE LIMITING as the size of response to an action potential is dependant on the number of primed vesicles
Involves Munc13 OPENING UP SYNTAXIN - this promotes SNARE complex assembly

Question 11

Action of Munc13 in priming?

Answer 11

Munc13 OPENS UP SYNTAXIN
VAMP can then enter complex
Allows vesicle to FULLY DOCK and promotes FULL SNARE COMPLEX assembly

Question 12

Role of GPCRs – what is meant by agonist-dependant plasticity?

Answer 12

GPCRs can regulate exocytosis in response to agonists therefore GPCRs display agonist-dependant activity

Question 13

Use of GPCR antagonist to measure secretion – method?

Answer 13

-Depolarise cells, increasing Calcium concentration
-Apply agonist (HISTAMINE) to activate GPCRs
»Capacitance of membrane increases when agonist added!

Histamine potentiator of exocytosis despite inhibition of Ca2+ entry, supporting idea that GPCRs can regulate exocytosis in presence of agonist

Question 14

Monitoring GPCR effects on vesicle docking and fusion – method and what was shown by overexpression of Munc13??

METHOD

Answer 14

• Vesicles loaded with flourescent dye
• Formation of Munc13 clusters at the plasma mem following stimulation of GPCRs – BUT IS INCREASING RECEP STIMULATION CAUSING MORE MUNC13 ACTIVITY

-Overexpression of Munc13, abolished Histamine-induced facilitation of exocytosis, but not the inhibition of VGCCs

Question 15

How does Munc13-1 prime vesicles for

release?

Answer 15

• Yeast 2 Hybrid screen showed N’ syntaxin binds to C’ Munc13-1
• Munc13-1 and syntaxin GST-fusion proteins bind in vitro
• Munc13-1 binds to 7S-core complex…..Regulate formation