Discovery

 SNARE discovery

Secretory mutants often referred to as Sec - mutants were created in yeast to identify genes involved in the secretory pathway much of this work was carried out by Novick and Schekman (1980).  None of the mutants could release secretory vesicles in addition some were temperature sensitive mutants. By this method 23 genes were identified and the mutants were named sec – followed by the number of the gene mutated i.e sec - 18 and classified into groups A to E based on where the block in the secretory pathway occurred when the gene was mutated.

All the sec genes were found to have at least one have mammalian homolog. In particular SEC18 mammalian homolog is NSF and SEC 17 gene required for transport from the ER to the golgi has several homologs called SNAPS alpha,beta and gamma. Rothman (1993) set out to investigate what other proteins were involved in the fusion machinery.  It was already identified that NSF was present at  membrane fusion and SNAP proteins had been purified as part of the fusion machinery. However when added to in vitro membrane system they could not initiate fusion. 

 Homegenised brain tissue was used to generate membrane extracts, chosen because of its dense synapse number meaning abundant fusion machinery and synaptic vesicles. To purify the other proteins of the fusion machinery, membrane extract was subject to affinity chromatography. Recombinant NSF with myc tag was produced by e.coli and attached to beads. Recombinant SNAPs were added to the column along with a form of non-hydrolasable ATP (added so that the NSF would not be active) and these were allowed to complex so that they could act as bait for other proteins of the fusion machinery. The membrane extracts were then added so that they could bind to the SNAP/NSF complex. Then hydrolysable ATP the type found naturally in cells was added to allow NSF to disassemble the complex in one version of the experiment. The components of the eludate from the column were then separated by SDS PAGE electrophoresis and the bands visualised and then sequenced. Some of the band sequences revealed NSF and SNAPs as expected, there were three bands not corresponding to known sequences for NSF or SNAP revealed no known protein. A second version of the experiment abandoned the addition of a hydrolysable ATP so that the whole complex could be isolated.

The three bands were identified and named individually but collectively they were collectively termed SNAREs:

• SNAP 25 – so named because it’s a 25 Kda protein.
• Synaptobrevin/VAMP
• Syntaxin – forms A and B were both found in the brain.

It was already known that SNAP-25 was located on the presynaptic terminal, synaptobrevin was known to be attached to the vesicle membrane and syntaxin known to be on the postsynaptic membrane. All three of these components sequences were found as part of the complex isolated by affinity chromatography was named the 20S complex consisting of SNAREs, SNAPS and NSF. NSF and SNAP complex is known as the 7S complex.

With this knowledge Rothman postulated:

• SNARES (SNAP 25, synaptobrevin/VAMP, syntaxin) form part of fusion apparatus for docking vesicle to the target membrane.
• And that because NSF and SNAP protein are also part of the complex they probably act in a similar manner. NSF uses ATP to drive fusion – NOW KNOWN TO BE WRONG
• Difference between constitutive and regulated secretion could be by the ability to regulate the fusion machinery. For instance secretion from neurons is stimulated by an increase in intracellular calcium.

Based on Rothmans experiments mechanisms of actions of the SNAREs have been modelled.

The first suggestion in part has now been proved, the SNAREs are localised as suggested and are involved in fusion. However NSF is now known not to be part of the fusion machinery but essential for SNARE recycling. The second suggestion has also been proven synaptotagmin is now known to be the calcium sensor that regulates neuroexocytosis.