Abstract
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 from 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.
Highlights
The molecular mechanisms driving movement of proteins between compartments have been unraveled, in part, by the development of cell-free assays that reconstitute various transport and fusion events
We present evidence that ␣-soluble NSF attachment proteins (SNAPs) is released from the membranes in a temperature- and time-dependent manner and that this release is mediated by the ATPase activity of N-ethylmaleimide-sensitive factor (NSF)
To gain further insight into the molecular mechanism involved in particle disassembly in a membrane environment, we studied the binding of recombinant SNAPs and NSF to endosomal membranes
Summary
The molecular mechanisms driving movement of proteins between compartments have been unraveled, in part, by the development of cell-free assays that reconstitute various transport and fusion events. NSF-dependent Release of ␣-SNAP Requires ATP Hydrolysis—It has been shown previously that ATP hydrolysis by NSF disassembles the SNARE complex [22, 25].
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