Dissecting the N-ethylmaleimide-sensitive factor: Required elemennts of the N and D1 domains

Chunxia Zhao, Elena A. Matveeva, Qiansheng Ren, Sidney W. Whiteheart

Research output: Contribution to journalArticlepeer-review

36 Scopus citations


N-Ethylmaleimide-sensitive factor (NSF) is a homo-hexameric member of the AAA+ (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP·SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg67 and Lys105, and the conserved residues in the central pore of NSF-D1 (Tyr296 and Gly298) are involved in SNAP·SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr373-Arg375), Sensor 2 (Glu440-Glu442), and Arginine Fingers (Arg385 and Arg388) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP·SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.

Original languageEnglish
Pages (from-to)761-772
Number of pages12
JournalJournal of Biological Chemistry
Issue number1
StatePublished - Jan 1 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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