title: Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.
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.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Cipriano
authors.forename: Jaemyeong
authors.initials: J
authors.lastname: Jung
authors.forename: Sandro
authors.initials: S
authors.lastname: Vivona
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Fenn
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
authors.forename: Zev
authors.initials: Z
authors.lastname: Bryant
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050600:SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050600:SNARE Proteins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins
keywords: AAA+ ATPase; ATPases; Exocytosis; Fluorescence; Mechanochemical Coupling; Membrane Fusion; Molecular Motors; Remodeling; Translocase; Unfoldase