L11 - SNAREs 1 - membrane fusion machinery

what are SNAREs?

proteins that mediate the fusion of vesicles with target membranes

what is membrane fusion?

• the process where two lipid bilayers merge to form one continuous membrane

what happens during membrane fusion?

• the membranes come close together - fusing proteins (SNAREs) pull them into contact

• the lipid bilayers mix and contents from one compartment are released into another

why is membrane fusion important?

• essential for moving materials within and out of cells

• crucial for secretion, communication and protein transport across neurons, endocrine, immune, digestive and all cellular systems

membrane fusion in nervous system

• synaptic vesicle fusion - neurotransmitter release

• communication between neurons and muscles

membrane fusion in endocrine and exocrine systems

• secretory granule fusion in pancreas

• hormones like insulin or digestive enzymes released

membrane fusion in immune and liver function

• secretion of serum proteins

• albumin from hepatocytes

• antibodies from plasma cells

membrane fusion in mucosal protection

• mucus secretion from epithelial mucosal cells

membrane fusion in general cell biology

• intracellular transport of proteins between organelles in all cells

• vesicle fusion between organelles

what are the 3 main approaches to identify the machinery of vesicle transport?

1. biochemical reconstitution

2. yeast genetics

3. cloning

what is biochemical reconstitution?

• recreates vesicle transport in a test tube using purified components to identify key proteins

what is yeast genetics?

• using mutant yeast to discover genes required for vesicle transport

what is cloning?

• involves copying and sequencing genes to study vesicle transport proteins in detail

explain biochemical reconstitution - Intra-Golgi transport assay (Rothman)

• mixed two Golgi fractions in a test tube

  Golgi Fraction 1 = inactive enzymes but a VSV-G (glycoprotein)

  Golgi Fraction 2 = active enzymes but no VSV-G

• if transport from one Golgi compartment to the other Golgi compartment occurred - the cargo moved from one Golgi to the next

• this showed that vesicles can mediate transport and fusion between Golgi compartments in vitro

• and specific proteins (SNAREs) are required for this process

what is NSF (N-ethylmaleimide - sensitive factor)?

• a crucial ATPase protein involved in membrane fusion processes (especially vesicle trafficking within cells)

what does NSF (N-ethylmaleimide - sensitive factor) do?

• helps with disassembly of SNARE complexes after vesicles fuse with membranes

• this allows SNAREs to be reused for further round of vesicle fusion

what does the combination of Golgi membranes + cytosol + ATP refer to?

• vesicle transport and membrane fusion

what does the increase in Golgi membranes + cytosol + ATP result in?

more radioactivity in the VSV-G protein

what is NEM?

a chemical called N-ethylmaleimide which inhibits proteins

what does the addition of NEM cause?

instead of an increase in radioactivity over time the addition of NEM caused inhibition of the reaction

• NEM inhibits intracellular trafficking and membrane fusion by blocking the function of NSF

• NEM inhibits NSF

what does NSF need to anchor it to the membrane?

SNAPs - Soluble NSF Attachment Protein

what happens to the SNAPs and NSF when membranes are salt washed?

• SNAPs get washed away

• NSF can no longer bind to the membranes (indirectly through SNAPs as they are gone)

what were yeast genetics used for?

• performed mutagenesis to find yeast mutants defective in secretion

• these mutants helped identify Sec genes involved in different steps of the secretory pathway

what happens with sec mutants like those with defects in Sec17 (a-SNAP) and Sec18 (NSF) genes?

• they start to accumulate many vesicles inside because vesicles can’t properly fuse with their target membranes

• this accumulation happens because the proteins needed for vesicle fusion are dysfunctional or missing

what do Sec1, Sec17 and Sec18 mutants have in common?

• they all have fusion blocked due to dysfunction or missing proteins

Sec 1 normal function

• Sec1 = SNARE binding protein - needed for SNARE complexes to work properly - and the SNARE complex is essential for driving fusion of membranes

what are Sec17 and Sec18?

Sec17 = encodes a-SNAP

Sec18 = encodes NSF

explain cloning as a way to identify the proteins involved in synaptic vesicle fusion

• pacific electric ray was used as its nerves have many synaptic vesicles

• its vesicles were purified and antibodies were made against them

• these antibodies were used to find and clone the genes for two key proteins:

  1. VAMP (on vesicles) - a V-SNARE

  2. Syntaxin (on target membranes) - a T-SNARE

     —> these two proteins are essential for vesicle fusion in neurons

what are clostridial neurotoxins and what do they do?

• clostridial neurotoxins = tetanus toxin + botulin toxin type B

  —> these are bacterial toxins

• they cleave (cut) VAMP (key vesicle SNARE protein)

• when VAMP is cleaved = vesicle fusion is blocked = neurotransmitters cannot be released from neurons

what 3 proteins make up the SNARE complex and what do they do?

VAMP

Syntaxin

SNAP-25

• the 3 proteins zip up together to pull membranes close and cause vesicle fusion

• after fusion - this complex disassembles when ATP is hydrolysed - requires proteins like NSF and a-SNAP

• —> so VAMP, Syntaxin and SNAP-25 are the key players in forming the SNARE complex that drives vesicle fusion

what is Rothman’s SNARE hypothesis?

1. different SNAREs exist for each transport step within the cell

2. SNAREs provide specificity - ensuring vesicles fuse only with the correct target membrane

3. SNAREs are sufficient to drive membrane fusion - bringing membranes together to fuse

how many SNAREs are there in the human genome?

• 38 SNAREs encoded in the human genome

• no SNAREs on mitochondria

explain the structure of the SNARE complex

• VAMP, Syntaxin and SNAP25 proteins zipper up together in a parallel coiled coil

• driving bilayer fusion by providing energy

What does SNARE zippering provide?

• energy to drive membrane fusion

• VAMP, Syntaxin and SNAP-25 zipper up together pulling vesicle and target membranes close enough to fuse

• bring two membrane together to fuse

what are R-SNAREs?

• a SNARE that contains an arginine residue

• usually found on vesicle membranes

• example - VAMP (synaptrobrevin)

—> most V-SNAREs are R-SNAREs

what are Q-SNAREs?

• a SNARE that contains a glutamine residue

• usually found on target membranes

• example - Syntaxin and SNAP-25

  —> most T-SNAREs are Q-SNAREs

what is the SNARE complex formed by interaction of?

• formed by interaction of 1 R-SNARE and 3 Q-SNAREs

• forming 4 helix bundle

• this tight assembly zippers up the membranes together and promotes fusion

• the 3Q:1R ration is conserved in all complexes

what do mutation of a Q/R cause?

inhibition of SNARE activity

• only get fusion with SNARE complexes which fit 3Q:1R ratio

what are the common features of SNARE proteins?

• SNARE motif or 1 coiled coil SNARE domain

• small proteins 14-40 kDa

• generally C-terminally anchored to membrane to drive membrane fusion

recombinant SNAREs can drive membrane fusion of purified liposomes what does this show?

• that SNARE proteins alone are sufficient to cause membrane fusion without other cellular factors