N-ETHYLMALEIMIDE-SENSITIVE FACTOR-DEPENDENT ALPHA-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 i ntracellular vesicle transport events, In vitro biochemical studies ha ve 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 re leased from the membranes in a temperature-and time-dependent manner a nd that this release is mediated by the ATPase activity of NSF, Our re sults indicate that NSF mutants in the first ATP binding domain comple tely abrogate alpha-SNAP release, whereas no inhibitory effect is obse rved with a mutant in the second ATP binding domain, Interestingly, ne ither beta-SNAP nor gamma-SNAP are released by the ATPase activity of NSF, indicating that these proteins are retained on the membranes by i nteractions that differ from those that retain alpha-SNAP, Although th e small Rab GTPases are known to play a role in SNARE complex assembly , our results indicate that these GTPases do not regulate the NSF-depe ndent release of alpha-SNAP.