SNAREs I Membrane fusion machinery Flashcards
Examples of membrane fusion happening in our body
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
Secretory vesicles can be visualized
by electron microscopy
3 main approaches were taken to identify the machinery of vesicle transport
1) Biochemical reconstitution.
2) Yeast Genetics.
3) Cloning.
What is Biochemical reconstitution
is aimed at identifying specific molecular reactions that operate at molecular-length scales
Example of Biochemical reconstitution:
Intra-Golgi transport assay
Intra-Golgi transport assay
‘Donor’ Golgi-containing fraction from VSV-infected 15B mutant
‘Acceptor’ Golgi-containing fraction from uninfected wild-type cells
The identification of NSF
N-ethylmaleimide inhibits reaction (alkalyting reagent).
Target purified and named N-ethylmaleimide Sensitive Factor (NSF 1988).
What is NSF?
NSF is an ATPase
What happens when membranes are salt washed
NSF can no longer bind to membranes.
Target purified and named SNAP Soluble NSF Attachment Protein (1990).
What was found in the isolation of sec mutants (NSF and α-SNAP)
SEC1 (SNARE binding protein), SEC17 encodes α-SNAP, SEC18 encodes NSF
What was cloned?
synaptic vesicle proteins (VAMP and Syntaxin)
Method of using Antibodies in cloning
Antibodies were raised against synaptic vesicles purified from electric rays.
The antibodies were then used to expression clone VAMP and Syntaxin.
What cleaves VAMP/synaptobrevin?
Clostridial neurotoxins tetanus and boutlinum B cleave VAMP / (synaptobrevin)
What was found in the biochemical purification of SNAREs
Found they could purify a large complex that dissembles
when ATP is hydrolyzed.
Rothman’s SNARE hypothesis
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!)
Crystal structure of the neuronal SNARE complex
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
Crystal structure provides
Mechanistic explanation of how SNAREs function
SNARE zippering though to provide?
energy to drive membrane fusion
Function of SNARE zippering
Bring two membranes together and fusing them is
energetically unfavorable
Where can SNAREs be divided into?
SNAREs can be divided into R and Q SNAREs
Q53, Q174 (SNAP25-N)
R56 (VAMP2)
Q226 (Syntaxin 1a)
Q and R ratio
3Qs to one R SNARE
conserved in all complexes
Mutation of a Q/R inhibits SNARE activity.
Do SNAREs provide the specificity of membrane fusion?
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)
Common features of SNARE proteins
Generally small 14-40kDa
All have at least 1 coiled-coil or SNARE motif
Generally C-terminally anchored
Are SNAREs the minimal fusion machinery?
Yes, put additional mechanisms for regulation
Recombinant SNAREs can drive
membrane fusion of purified liposomes
What does NSF do after fusion?
NSF recycles the SNAREs after fusion
What is NSF not required for?
NSF not required for the fusion step!
What does NSF act on?
NSF acts on cis-SNARE complex
