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J Biol Chem. 2013 Aug 9;288(32):23436-45. doi: 10.1074/jbc.M113.476705. Epub 2013 Jun 17.

Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.

Author information

1
Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

Abstract

SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

KEYWORDS:

AAA+ ATPase; ATPases; Exocytosis; Fluorescence; Mechanochemical Coupling; Membrane Fusion; Molecular Motors; Remodeling; Translocase; Unfoldase

PMID:
23775070
PMCID:
PMC4520572
DOI:
10.1074/jbc.M113.476705
[Indexed for MEDLINE]
Free PMC Article

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