Disassembly of membrane-associated NSF20S complexes is slow relative to vesicle fusion and is Ca2+-independent

N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble N SF attachment protein (alpha-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S pro tein complex, However, their precise function, relative to fusion itself, i s not clear, Using a W-activated crosslinking approach, we have measured th e rate at which a single round of NSF-driven ATP hydrolysis leads to 20S co mplex disassembly within synaptic membranes, Although this rate is substant ially faster than previous estimates of NSF-dependent ATP hydrolysis, it re mains much lower than published rates for fusion of synaptic vesicles. Furt hermore, the stability of 20S complexes is unaffected by Ca2+ at concentrat ions that elicit rapid membrane fusion, We conclude that the ATPase activit y of NSF does not contribute directly to vesicle fusion, but more likely pl ays an earlier role in the synaptic vesicle cycle.