SNAREs I Membrane fusion machinery Flashcards

1
Q

Examples of membrane fusion happening in our body

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Secretory vesicles can be visualized

A

by electron microscopy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is Biochemical reconstitution

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Example of Biochemical reconstitution:

A

Intra-Golgi transport assay

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Intra-Golgi transport assay

A

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

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

The identification of NSF

A

N-ethylmaleimide inhibits reaction (alkalyting reagent).

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is NSF?

A

NSF is an ATPase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What happens when membranes are salt washed

A

NSF can no longer bind to membranes.

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What was cloned?

A

synaptic vesicle proteins (VAMP and Syntaxin)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Method of using Antibodies in cloning

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What cleaves VAMP/synaptobrevin?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What was found in the biochemical purification of SNAREs

A

Found they could purify a large complex that dissembles

when ATP is hydrolyzed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Rothman’s SNARE hypothesis

A

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!)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Crystal structure of the neuronal SNARE complex

A

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

17
Q

Crystal structure provides

A

Mechanistic explanation of how SNAREs function

18
Q

SNARE zippering though to provide?

A

energy to drive membrane fusion

19
Q

Function of SNARE zippering

A

Bring two membranes together and fusing them is

energetically unfavorable

20
Q

Where can SNAREs be divided into?

A

SNAREs can be divided into R and Q SNAREs

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

21
Q

Q and R ratio

A

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

22
Q

Do SNAREs provide the specificity of membrane fusion?

A

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)

23
Q

Common features of SNARE proteins

A

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

24
Q

Are SNAREs the minimal fusion machinery?

A

Yes, put additional mechanisms for regulation

25
Q

Recombinant SNAREs can drive

A

membrane fusion of purified liposomes

26
Q

What does NSF do after fusion?

A

NSF recycles the SNAREs after fusion

27
Q

What is NSF not required for?

A

NSF not required for the fusion step!

28
Q

What does NSF act on?

A

NSF acts on cis-SNARE complex