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.

SUBMITTER: Cipriano DJ 

PROVIDER: S-EPMC4520572 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature