SNAREs

List the physiological systems which require membrane fusion

Discuss the experimental evidence for the role of SNAREs in membrane fusion.

Describe the key features of a SNARE molecule.

Discuss why SNARE molecules are required to drive membrane fusion.

amino acids found in high abundance in signals required to trigger active transport in the nucleus

pro, lys, arg

List some essential mechanisms carried out by membrane fusion 

• 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

Which 2 peptide hormones are transported through secretory granule fusion ie exocytosis?

insulin and glucagon

How were secretory vesicles first identified experimentally? What was understood at this stage?

using electron microscope + stimulation of cells showed mvt of granules implying their role in the stimulated process

mechanism vas visualised but not molecularly understood

What were 3 approaches taken to identify the machinery of vesicle transport, bringing vesicle understanding from microscopic anatomy to that of the molecular machinery of vesicle fusion?

1. Biochemical reconstitution.

2. Yeast Genetics.

3. Cloning.

Describe biochemical reconstitution

experimental technique where a complex biological process is recreated in vitro (outside of a living cell) using a minimal set of purified components

Aim of biochemical reconstitution

identify the necessary molecules and understand the fundamental mechanisms of a process by isolating and testing the function of specific molecules by reassembling them under controlled conditions

Explain the intra-golgi transport assay

• cell-free, only donor golgi with labelled mutant that cannot be modified mixed + acceptor golgi with WT + cytosol and ATP

• labelled cargo will be modified by acceptor membranes

• quantify transport rates, identify necessary components (like specific proteins and cytosolic factors), and test how different conditions affect the process

What was biochemical reconstitution used before looking at membrane fusion? Why was there scepticism when looking at this technique for membrane fusion?

mechanisms of ATP synthesis, DNA replication, RNA transcription, protein synthesis, the genetic code

microscopy showed membranes in very close proximity - believed that if this homogenisation was destroyed then transport would no longer happen

How was biochemical reconstitution used to understand membrane fusion?

intra-golgi transport assay showed necessary conditions to the process and what affects it eg temperature, presence of specific molecules etc 

What does NSF stand for and what is its role?

N-ethylmaleimide Sensitive Factor

ATPase so hydrolises

What effect does N-ethylmaleimide have on a NSF? What is it therefore termed?

inhibits it

alkylating reagent

What does N-ethylmaleimide bind to? What does this lead to?

N-ethylmaleimide Sensitive Factor ie NSF

inhibition of NSF

What allows NSF to bind to membranes, allowing the binding of N-ethylmaleimide to inhibit a reaction?

SNAP ie soluble NSF attachment protein

How was SNAP found?

When washing membranes with salt wash - SNAP target was rinsed away and identified at the target of NSF, allowing their attachment to the membrane

What effect do SNAREs have on SNAP function? Where does the term SNARE come from?

SNAREs are proteins that allow SNAP to bind to membranes ie to allow N-ethylmaleimide to bind NSF ATPases to inhibit reactions

SNAP + REceptor

What is the role of NSF in membrane fusion? 

• part of a molecular machinery that builds SNAREs = membrane fusion

• NSF uses ATP hydrolysis to disassemble it, resetting the SNARE proteins so they can be used again

What does SNARE stand for?

Soluble NSF Attachment Protein Receptor

How does SNAP contribute to the disassembly of SNAREs?

recruits NSF to membrane, NSF then hydrolyses SNARE complex, disassociating it to allow for their recycling

In what manner does NSF bind membranes?

cycles on and off membranes in an ATP dependent manner at SNAP receptors, making up the 20 S fusion particle

What makes up the 20 S fusion particle ?

NSF ATPase + SNAP proteins + SNARE complex 

When was the first time that it was realised that the same machinery was conserved between yeast and man?

1980 by Randy Schekman when looking at sec, NSF and SNAPs

What are alpha SNAP and sec 17 protein? What distinguishes them?

proteins binding with SNARE complexes + recruiting motor proteins NSF/Sec18

alpha SNAP and NSF in mammals // sec 17 and sec18 in yeast

What is the role of NSF/sec18?

along with alpha SNPA/sec 17, to regulate (sometimes assembly)/disassembly of cis-SNARE complexes by inducing (zippering) hydrolysis = recycling of SNARE complexes

Which gene encodes both sec 17 proteins (in yeast) and alpha SNAP (in mammals)?

sec 17 gene 

Which gene encodes NSF?

sec 18 gene

What would a mutation in sec17 or sec 18 genes inhibit?

no assembly and disassembly of SNARE proteins bc non functional alpha SNAP or NSF respectively = no membrane fusion = no vesicular transport = no exchange of proteins between ER and golgi = no proteins made + accumulation of transport vesicles

What is the role of sec1 proteins?

encode Munc18 proteins that protect trans-SNARE complex from sec17/18 proteins ie alpha SNAP and NSF - preventing their disassembly and allowing it to complete assembly and mediate fusion

What does sec 1 encode in mammals?

Munc18 proteins

What are sec 1, sec 17, sec 18?

genes encoding proteins involved in membrane fusion by allowing the association or disassembly of complexes forming with SNAREs and vesicles forming and fusing

Why are pacific electric rays commonly used as models?

large neurons so good for imaging

How were clostridial neurotoxins tetanus and botulinum B used to infer the role of VAMP?

purified vesicles were taken and exposed to these toxins - showed rigidity and paralysis as VAMP disappeared → evidence VAMP is involved in membrane fusion

How were VAMP and Syntaxin discovered experimentally? 

through cloning of synaptic vesicles in electric rays

What do clostridial neurotoxins tetanus and botulinum B cause? 

paralysis and rigidity 

What are the 3 key snare molecule identified?

VAMP, SNAP25 and syntaxin

How were the components of SNARE complexes first purified?

biochemical complementation assay - NEM treated cytosol ie NSF inactive - allowed identification, isolation and purification of non-NEM sensitive components (Jim Rothman)

What were the 4 ideas making up Rothman’s SNARE hypothesis?

1) SNAREs for each transport step within the cell.

2) SNAREs should provide specificity to vesicle transport. (not entirely true)

3) SNAREs should be sufficient to drive lipid bilayer fusion.

4) Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this bit is wrong!)

Breaking down Rothman’s SNARE hypothesis, is the first part true, ie Are there SNAREs for every transport step? What types of experimental techniques were used for this research?

yes, validated! cloning trafficking material

How many different SNAREs are encoded in the human genome? What are they involved in?

38, various transport steps and fusion reactions 

How was it determined that SNARE complexes are formed by zippering in a parallel coiled coil?

through crystallisation of the purified SNARE

How does a SNARE complex form?

VAMP, Syntaxin and SNAP25 come together and zipper in a parallel coiled coil

What provides energy necessary to drive membrane fusion?

the zippering of the snare complex itself

Is bringing two membranes together and fusing them energetically favourable?

no! zippering of snare complex provides the necessary energy for membrane fusion

What distinguishes trans from cis SNARE complexes?

trans complex formed when SNARE proteins from different membranes pair up, fusing the membranes // snare proteins are on the same, now-fused membrane

What distinguishes R from Q SNAREs?

R snares contain an R residue, amino acid Arginine // Q ones contain a glutamine amino acid residue

Among VAMP, Syntaxin and SNAP25, which are Q and which are R SNAREs?

VAMP is R, syntaxin and SNAP25 are Q

What type of SNAREs do R-SNAREs typically act as?

arginine residue containing SNAREs usually act as v-SNAREs ie vesicle associated SNAREs

What type of SNAREs do Q-SNAREs typically act as?

glutamine residue containing SNAREs usually act as t-SNAREs ie target-membrane associated SNAREs

What is the ratio of Q to R SNAREs maintained in all complexes in vivo?

3Q:1R

How would a mutation in Q/R residues in SNAREs affect SNARE activity?

inhibits it

What does the following figure represent and inform on Q/R ratios maintained in all complexes in vivo?

shows various snare complexes and the snares making them up - always a 3Q to 1 R ratio maintained, no matter the different isoforms of VAMP, Syntaxin and SNAP25 involved

Breaking down Rothman’s SNARE hypothesis, is the second part true, ie Do SNAREs provide the specificity of membrane fusion? 

half and half - they definitely contribute to specificity, bc even though snares show some promiscuity they mainly interact with snares from appropriate membranes HOWEVER lots of additional machinery contribute to specificty (i.e. rabs/ coat proteins and tethers) so maybe can’t say they PROVIDE the specificity

What are common features of SNARE proteins whether they have Q or R residues?

• generally small 14 to 40 kDa

• all have at least 1 coiled coil/SNARE motif

• tm domain

• c terminally anchored

What type of SNARE are Qb, Qc and Qbc SNAREs?

SNAP 25 - subfamilies of Q SNARE dependent on their location in the 4 helix bundle that SNARE complexes form

Breaking down Rothman’s SNARE hypothesis, is the third part true, ie Are SNAREs sufficient to drive lipid bilayer fusion? Which experimental technique showed this?

yes ! shown to be the minimal fusion machinery 

TIRF microscopy 

Breaking down Rothman’s SNARE hypothesis, is the fourth part true, ie do NSF and ATP hydrolysis catalyse membrane fusion?

WRONG 

Can recombinant SNAREs fuse membranes?

yes!

What is NSF role simply in fusion?

recycles SNAREs

Via which structure do SNAREs zipper up?

via their coiled-coil domains

How are SNARE proteins generally anchored?

thanks to their C-terminal

In which fusion steps in the endocytic/biosynthetic pathways are SNAREs required?

ALL fusion steps

Which SNARE complex does NSF specifically act on?

cis-SNARE ie when fusion has occurred and SNAREs are all in the same membrane, following membrane fusion of trans SNAREs

How is NSF implicated in fusion step of membrane fusion?

isn’t! simply recycles SNAREs after fusion so essential for trafficking but not for the fusion step

Are other proteins needed alongside recombinant SNAREs to drive membrane fusion of purified liposomes? 

yes! SNAREs alone can lead to membrane fusion but without calcium and Habc domains eg, much slower and would be much too slow for NTssion eg + proteins like munc 18 and complexin will provide specificity and control of fusion, NSF finally allowing resetting/recycling of SNAREs

List proteins needed in a purified liposome for successful, precise, specific and rapid membrane fusion to occur

• SNAREs for membrane fusion

• calcium for speed

• munc18 for specificity

• complexin/synaptotagmin for control

• NSF for resetting and recycling

• open Habc domain

How does calcium affect the fusion rate of purified membranes only in the presence of SNAREs?

increases by about 30 to 50 fold

What is the role of the Habc domain on syntaxin/Qa SNAREs in membrane fusion ? 

regulates assembly of SNARE complex, acting as a lid to maintain the complex in an inactive closed conformation - inhibits unwanted fusion events. opens to allow SNARE binding

Qa SNARE

syntaxin

Qb, Qc, Qbc SNARE

SNAP25

R SNARE

VAMP

Where is the regulatory Habc domain typically found in a SNARE complex? What can this domain interact with?

at the N terminus of syntaxins/Q a SNAREs

Munc18/nsec1, ubiquitin, other SNARE domains for formation of SNARE complex

Would membrane fusion happen in the absence of calcium and presence of a closed Habc domain? 

no - although snares are sufficient to fusion, calcium speeds it and Habc will typically inhibit syntaxin from binding the other snares to form the snare complex