Soluble N-ethylmaleimide-Sensitive Factor Attachment Proteins (SNAP, or Sec17p in yeast) are a family of cytosolic adaptor proteins involved in vesicular fusion at membranes during intracellular transport and exocytosis. SNAPs interact with proteins of the SNARE complex and NSF to play a key role in recycling the components of the fusion complex. SNAPs are involved in the priming of the vesicle fusion complex during assembly, as well as in the disassembly following a vesicle fusion event. Following membrane fusion, the tethering SNARE proteins complex disassembles in response to steric changes originating from the ATPase NSF. The energy provided by NSF is transferred throughout the SNARE complex and SNAP, allowing the proteins to untangle, and recycled for future fusion events. Mammals have three SNAP genes: α-SNAP, β-SNAP, and γ-SNAP. α- and γ-SNAP are expressed throughout the body, while β-SNAP is specific to the brain. The yeast homolog of the human SNAP is Sec17.
Function The function of SNAP proteins have been primarily related to the role which the play in the assemble and disassembly of SNARE complex required for vesicle fusion events. According to the SNARE hypothesis developed in the early 1990s, SNAP protein are localized to the membranes and are central in mediating Ca2+ dependent vesicle fusion at these sites. SNAPs associate with the proteins of the SNARE (SNAP REceptor) complex, a class of type II integral membrane protein, as well as the ATPase NSF, largely based on electrostatic interactions. The interaction of the SNAPs with SNAREs takes place before interaction of the complex with NSF (Sec18 in yeast) suggesting a sequence for the priming assembly may be necessary. The assembled complex which includes SNAP, SNARE, and NSF is known as the 20S complex. Some of the first proteins identified as the receptors of SNAPs were syntaxin 1, SNAP-25 (synaptosome associated protein, 25kDa), and VAMP (synaptobrevin). These proteins contain transmembrane regions that can be found in both intracellular vesicles and as part of extracellular trafficking machinery. Figure 1 shows interactions of the vesicular and membrane SNARE proteins with NSF and SNAP in the assembly, fusion, and disassembly process that accompanies vesicle fusion events. Initial binding of NSF to SNAP been is likely related to interactions of the 63 N-terminal and 37 C-terminal amino acid residues of SNAP with NSF protein. The interaction with SNAP stimulates the ATPase activity of the NSF when assembled into the 20S complex, and ultimately leads to ATP hydrolysis that result in the disruption of the heterooligomeric complex. This has the potential to reduce or block synaptic transmission, ultimately leading to the loss of signaling downstream. Further information on this is included in the toxicology section below. While assembly of the complex can take place under only conditions where a components and a membrane is present, disassembly requires that NSF can hydrolyze ATP. Use of chelating agents, non-hydrolysable analogues of GTP, or application of an alkylating agent N-ethylmaleimide (NEM), therefore, has been used to demonstrate prevention of vesicle fusion in vitro. Blocking the assembly of the 20S complex also prevents the ATP-hydrolysis reaction from taking place at NSF.
Limitations of the Original SNARE Theory of Vesicle Fusion
The SNARE theory of vesicle fusion, describes the action mechanism of SNAREs, SNAP, and NSF, but does not completely explain all known vesicle fusion related kinetics. The theory was first put forth by James Rothman and co-workers starting in the early 1990s and predicted that SNAPs and NSF recognized paired vesicle-SNARE (v-SNARE)/ target-SNARE (t-SNARE) complexes at membranes and bound to them thus creating the 20S complex. These complex form similar structures for both synaptic and vacuolar systems including the Golgi transport. Data generated experimentally in recent years lead some to question the completeness of the model. Although it was known since the 1960s that Ca2+ influx was responsible for synaptic signaling, a collaboration in 1992 between Thomas Südhof and Reinhardt Jahn tied the link between calcium, SNARE complexes and synaptic signaling, suggesting that vesicle fusion events were not rate limited by the SNARE complex formation as previously thought. At the time, the SNARE complex model could not account for the rapid release of neurotransmitters into synaptic clefts, as the complex disassociation and recycling was thought to be rate limiting for further vesicle fusion. Further studies demonstrated that the ATP hydrolysis step occurs prior to a calcium ion mediated fusion event, and thus revealing, that SNAP and NSF proteins initiate disassembly the 20S complex before the docking event takes place directly at the membrane. The existence of these ATP primed vesicles for fusion at the pre-synaptic membrane is facilitated by the interactions of SNAP and NSF. It is now understood that the 20S complex does not disassociate immediately following ATP hydrolysis, but rather remains tethered until intracellular Ca2+ achieve significantly high levels to facilitate docking. A depolarizing current that leads to the opening of voltage dependent ion channels permits the influx of Ca2+ into the cell where the molecular clamp protein (a SNARE) called synaptotagmin acts in a Ca2+ sensitive manner to facilitate fusion of the vesicle to the membrane up to a rate of one vesicle per 100us. The exocytosis of neurotransmitters as regulated by Ca2+ therefore, has faster kinetics than would be possible by the SNARE-recycling model alone. Figure 1 summarizes the updated model of the SNARE hypothesis.
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