SNAREs I Membrane fusion machinery

Question 1

Examples of membrane fusion happening in our body

Answer 1

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)

Intracellular transport of proteins between organelles in all of your cells

Question 2

Secretory vesicles can be visualized

Answer 2

by electron microscopy

Question 3

3 main approaches were taken to identify the machinery of vesicle transport

Answer 3

1) Biochemical reconstitution.
2) Yeast Genetics.
3) Cloning.

Question 4

What is Biochemical reconstitution

Answer 4

is aimed at identifying specific molecular reactions that operate at molecular-length scales

Question 5

Example of Biochemical reconstitution:

Answer 5

Intra-Golgi transport assay

Question 6

Intra-Golgi transport assay

Answer 6

‘Donor’ Golgi-containing fraction from VSV-infected 15B mutant

‘Acceptor’ Golgi-containing fraction from uninfected wild-type cells

Question 7

The identification of NSF

Answer 7

N-ethylmaleimide inhibits reaction (alkalyting reagent).

Target purified and named N-ethylmaleimide Sensitive Factor (NSF 1988).

Question 8

What is NSF?

Answer 8

NSF is an ATPase

Question 9

What happens when membranes are salt washed

Answer 9

NSF can no longer bind to membranes.

Target purified and named SNAP Soluble NSF Attachment Protein (1990).

Question 10

What was found in the isolation of sec mutants (NSF and α-SNAP)

Answer 10

SEC1 (SNARE binding protein), SEC17 encodes α-SNAP, SEC18 encodes NSF

Question 11

What was cloned?

Answer 11

synaptic vesicle proteins (VAMP and Syntaxin)

Question 12

Method of using Antibodies in cloning

Answer 12

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

Question 13

What cleaves VAMP/synaptobrevin?

Answer 13

Clostridial neurotoxins tetanus and boutlinum B cleave VAMP / (synaptobrevin)

Question 14

What was found in the biochemical purification of SNAREs

Answer 14

Found they could purify a large complex that dissembles

when ATP is hydrolyzed.

Question 15

Rothman’s SNARE hypothesis

Answer 15

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 bit is wrong!)

Question 16

Crystal structure of the neuronal SNARE complex

Answer 16

VAMP on vesicle membrane
Syntaxin and SNAP25 on the target membrane
SNAREs zipper in a parallel coiled coil

they bind and drive fusion bringing two membranes together

Question 17

Crystal structure provides

Answer 17

Mechanistic explanation of how SNAREs function

Question 18

SNARE zippering though to provide?

Answer 18

energy to drive membrane fusion

Question 19

Function of SNARE zippering

Answer 19

Bring two membranes together and fusing them is

energetically unfavorable

Question 20

Where can SNAREs be divided into?

Answer 20

SNAREs can be divided into R and Q SNAREs

Q53, Q174 (SNAP25-N)
R56 (VAMP2)
Q226 (Syntaxin 1a)

Question 21

Q and R ratio

Answer 21

3Qs to one R SNARE
conserved in all complexes
Mutation of a Q/R inhibits SNARE activity.

Question 22

Do SNAREs provide the specificity of membrane fusion?

Answer 22

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 specificty (i.e. rabs/ coat proteins and tethers)

Question 23

Common features of SNARE proteins

Answer 23

Generally small 14-40kDa
All have at least 1 coiled-coil or SNARE motif
Generally C-terminally anchored

Question 24

Are SNAREs the minimal fusion machinery?

Answer 24

Yes, put additional mechanisms for regulation

Question 25

Recombinant SNAREs can drive

Answer 25

membrane fusion of purified liposomes

Question 26

What does NSF do after fusion?

Answer 26

NSF recycles the SNAREs after fusion

Question 27

What is NSF not required for?

Answer 27

NSF not required for the fusion step!

Question 28

What does NSF act on?

Answer 28

NSF acts on cis-SNARE complex