title: Analysis of NSF mutants reveals residues involved in SNAP binding and ATPase stimulation.
abstract: N-Ethylmaleimide-sensitive fusion protein (NSF) and its yeast orthologue, Sec18, are cytoplasmic AAA(+) ATPases required for most intracellular membrane fusion events. The primary function of NSF is thought to be the disassembly of cis-SNARE complexes, thus allowing trans-SNARE complex formation and subsequent membrane fusion. The importance of NSF/Sec18 in intracellular membrane traffic in vivo is highlighted by the inhibition of neurotransmission in Drosophila comatose (NSF) mutants and of constitutive secretion in yeast sec18 mutants. However, the underlying biochemical defects in these mutant proteins are largely unknown. Here, we identify the sec18-1 mutation as a G89D substitution in the N domain of Sec18p. This mutation results in an inhibition of the mutant protein's ability to bind to Sec17p (yeast alpha-SNAP). In contrast, engineering the comatose(st53)() mutation (S483L) into mammalian NSF (S491L) has no effect on alpha-SNAP binding. Instead, the stimulation of ATPase activity by alpha-SNAP required for wild-type NSF to disassemble SNARE complexes does not occur in the mutant NSF(st53) protein. This biochemical phenotype predicts a dominant negative effect, which was confirmed by engineering the st53 mutation into Sec18 (A505L), resulting in a dominant lethal phenotype in vivo. These findings suggest a biochemical basis for the block in membrane fusion observed in the mutant organisms. Furthermore, the mutants characterized here define key residues involved in two essential, but mechanistically distinct, biochemical functions of NSF: SNAP binding and SNAP-dependent ATPase stimulation.
authors.affiliation: The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
authors.forename: William G C
authors.initials: WG
authors.lastname: Horsnell
authors.forename: Gregor J
authors.initials: GJ
authors.lastname: Steel
authors.forename: Alan
authors.initials: A
authors.lastname: Morgan
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D004331:Drosophila melanogaster / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D005998:Glycine / Q000235:genetics; D008561:Membrane Fusion / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D052066:N-Ethylmaleimide-Sensitive Proteins; D051381:Rats; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C498732:comt protein, Drosophila; D001224:Aspartic Acid; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide; D005998:Glycine