Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca(2+)-independent.
N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (alpha)-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca(2+) at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.
Swanton
E
E
Division of Biochemistry, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
Bishop
N
N
Sheehan
J
J
High
S
S
Woodman
P
P
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D052066:N-Ethylmaleimide-Sensitive Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013552:Swine; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D008274:Magnesium; D002118:Calcium