Lecture 10 - SNAREs 1: Membrane fusion machinery

Question 1

Describe how membrane fusion occurs all the time

Answer 1

• Synaptic vesicle fusion (communication between neurons and muscles
• Secretory granule fusion (endocrine & exocrine pancreas)
• Secretion of serum protein (albumin from hepatocytes and antibodies from plasma cells)
• Mucus secretion (epithelial mucosal cells
• Intracellular transport of proteins between organelles in all of your cells

Question 2

What can secretory vesicles be visualized by?

Answer 2

electron microscopy - first developed in 1938

Question 3

What 3 ways allow you to investigate the machinery of vesicle transport?

Answer 3

1. Biochemical reconstitution
2. Yeast Genetics
3. Cloning

Question 4

What can you use as an assay for biochemical reconstitution to determine the machinery of vesicle transport?

Answer 4

Intra-Golgi transport

Question 5

Describe the identification of NSF (N-ethylmaleimide Sensitive Factor)

Answer 5

• N-ethylmaleimide (NEM) - alkylating agent that inhibits the activity of NSF, which is an ATPase involved in the disassembly of SNARE complexes during vesicle fusion.
• When membranes are salt washed NSF can no longer bind to membranes. Target purified and named SNAP (soluble NSF attachment protein)
• SNAPs bind to the membranes via hypothetical proteins called SNAP Receptors (SNAREs)

Question 6

What does SEC17 encode?

Answer 6

a-SNAP

Question 7

What does SEC18 encode?

Answer 7

NSF

Question 8

Explain how machinery is conserved between yeast and human

Answer 8

• same machinery used
• Yeast Sec18 works in Golgi transport assay
• Used Sec17 to confirm a-SNAP required for transport
• When secretion blocked, cells changed density, mutants separated on density gradient based on floor polish (23 complementation groups).

Question 9

Explain the process of cloning synaptic vesicle proteins

Answer 9

Antibodies were raised against synaptic vesicles purified from electric rays. The antibodies were then used to expression clone VAMP and Synataxin.

Question 10

What cleaves VAMP/(synaptobrevin)?

Answer 10

Clostridial neurotoxins tetanus and botulinum B cleaves VAMP/(synaptobrevin)
- Botulism causes paralysis. Looking for target. Checked synaptic vesicles. Zinc protease. Tetanus toxin cause lockjaw cleave snare machinery

• VAMP a good candidate for being a component of membrane machinery

Question 11

Describe the biochemical purification of SNAREs

Answer 11

Found they could purify a large complex that dissembles when ATP is hydrolyses

Question 12

What is Rothman’s SNARE hypothesis?

Answer 12

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 13

Are there SNAREs for every transport step?

Answer 13

38 SNAREs encoded in the human genome. Involved in various transport steps and fusion reactions. Few more SNAREs being identified but most found.

Question 14

Describe the crystal structure of the neuronal SNARE complex

Answer 14

SNAREs zipper in a parallel coiled coil
VAMP + SNAP25 + Syntaxin

Question 15

What is thought to provide energy to drive membrane fusion?

Answer 15

SNARE zippering is thought to provide energy to drive membrane fusion.
- bring 2 membranes together and fusing them is energetically unfavourable

Question 16

What can SNAREs be divided into?

Answer 16

Q & R SNAREs

Question 17

Describe the distribution of Q:R SNAREs

Answer 17

3 Qs to 1 R SNARE
- 3Q:1R ratio conserved in al complexes
Mutation of a Q/R SNARE inhibits SNARE activity

Question 18

Do SNAREs provide the specificity of membrane fusion?

Answer 18

• Only get fusion with SNARE complexes which fit 3Q:1R ratio.
• SNAREs show some promiscuity but, on the whole, they predominantly interact with SNAREs from the appropriate membranes.
• Lots of additional machinery contribute to specificity (i.e. rabs/coat proteins and tethers)

Question 19

Describe common features of SNARE proteins

Answer 19

• generally small 14-40 kDa
• all have at least 1 coiled-coil or SNARE motif
• generally C-terminally anchored

Question 20

Explain how Recombinant SNAREs can drive membrane fusion of purified liposomes

Answer 20

Calcium increased fusion rate about 30-50 fold. However, removal of SNAREs or cleavage of SNAREs with toxins resulted in no fusion when Ca added.

Question 21

What does NSF do?

Answer 21

recycles the SNAREs after fusion
- NSF not required for the fusion step
- The structure of the SNARE/NSF complex has been solved

Question 22

Summarise SNAREs

Answer 22

1) SNAREs are required for all fusion steps in the endocytic/biosynthetic pathways
2) SNAREs are generally small C-terminal anchored proteins
3) SNAREs zipper up via their coiled-coil domains
4) SNAREs provide some of the specificity for membrane fusion
5) SNAREs can be classified into Q & R SNAREs
6) Most SNARE complexes contain a ratio of 1Q:1R SNAREs
7) Recombinant SNAREs (synthetically produced SNAREs)can fuse membranes
8) NSF acts to recycle SNAREs after fusion

Question 23

What does SNARE stand for?

Answer 23

Soluble N-ethylmaleimide-sensitive Factor Attachment Receptor