N-Ethylmaleimide-sensitive factor-dependent α-SNAP release, an early event in the docking/fusion process, is not regulated by Rab GTPases

The N-ethylmaleimide-sensitive factor (NSF) is required for multiple intracellular vesicle transport events, In vitro biochemical studies have demonstrated that NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors form a 20 S particle, This complex is disassembled by the ATPase activity of NSF, We have studied particle disassembly in a membrane environment by examining the binding of recombinant SNAPs and NSF to endosomal membranes, We present evidence that alpha-SNAP is released from the membranes in a temperature-and time-dependent manner and that this release is mediated by the ATPase activity of NSF, Our results indicate that NSF mutants in the first ATP binding domain completely abrogate alpha-SNAP release, whereas no inhibitory effect is observed with a mutant in the second ATP binding domain, Interestingly, neither beta-SNAP nor gamma-SNAP are released by the ATPase activity of NSF, indicating that these proteins are retained on the membranes by interactions that differ from those that retain alpha-SNAP, Although the small Rab GTPases are known to play a role in SNARE complex assembly, our results indicate that these GTPases do not regulate the NSF-dependent release of alpha-SNAP.