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 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.
Colombo
M I
MI
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gelberman
S C
SC
Whiteheart
S W
SW
Stahl
P D
PD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013696:Temperature; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins