L10 - SNAREs 1

Question 1

What is a SNARE protein?

Answer 1

SNARE proteins are molecular motors that drive the biological fusion of two membranes

Question 2

What are the 3 main approaches taken to identify machinery of vesicle transport?(3)

Answer 2

1) Biochemical reconstitution
2) Yeast genetics
3) Cloning

Question 3

What is a NSF?

Answer 3

An enzyme

Question 4

What are examples of membrane fusion in the body? (4)

Answer 4

Synaptic vesicles fusion (communication between neurons
and muscles)

Secretory granule fusion (endocrine and exocrine pancreas)

Secretion of serum proteins (albumin from hepatocytes and
antibodies from plasma cells)

Mucus secretion (epithelial mucosal cells)

Question 5

How does NSF cycle on and off in membranes?

Answer 5

Does this in an ATP dependant manner

Question 6

How do we show prescience of NSF/SNAP?

Answer 6

We add ATP inhibitors, NSF binds to SNAP and reaction is killed without ATP

Question 7

SEC mutant were isolated what do these mutants do?
SEC18
SEC17

Answer 7

-Yeast SEC18 works in Golgi transport and encodes NSF
-Used SEC17 to confirm alpha SNAP required for transport

Question 8

What kind of trafficking was NSF found to be required in?

Answer 8

All eukaryotic

Question 9

Which proteins were found in synaptic vesicles? (2)

Answer 9

VAMP and Syntaxin

Question 10

What was Rothman’s SNARE hypothesis? (4)

Answer 10

1) SNAREs for each transport step within the cell
2) SNAREs should provide specificity to vesicle transport
3) SNAREs should be sufficient to drive lipid bilayer fusion
4) Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this is wrong)

Question 11

What are the main 3 SNAREs for transport and fusion? (3)

Answer 11

-VAMP
-Syntaxin
-SNAP25

Question 12

What is though to provide energy and drive membrane fusion?

Answer 12

SNARE zippering

Question 13

What forms SNAP25? (3)

Answer 13

-Syntaxin, VAMP and two coils form

Question 14

What type of SNARE is:
VAMP -
Syntaxin -

Answer 14

VAMP - Q SNARE
SYntaxin - R SNARE

Question 15

What is the ratio of Q to R SNAREs?

Answer 15

-There are 3 Q SNAREs to 1 R SNARE. This ratio is conserved in all complexes

Question 16

Do SNAREs provide specificity of membrane fusion? (3)

Answer 16

-Only get fusion with SNARE complexes that have the 3:1 ratio
-SNARE domains have preferences for interaction
-Lots of additional machinery contribute to specificity

Question 17

What are common features of SNARE proteins? (4)

Answer 17

-Generally small 14-40kDa
-All have at least 1 coiled-coil or SNARE motif
-C-terminally anchored
-N terminus of these proteins usually in cytoplasm but C terminus on membrane

Question 18

What can drive membrane fusion of purified liposomes?

Answer 18

Recombinants SNAREs

Question 19

What recycles after SNARE fusion?

Answer 19

NSF
(NSF acts on cis-SNARE complex)