EACH DOMAIN OF THE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN CONTRIBUTES TO ITS TRANSPORT ACTIVITY

N-Ethylmaleimide-sensitive fusion protein (NSF) has been shown to be i nvolved in numerous intracellular transport events. In an effort to un derstand the basic mechanism of NSF in vesicle-target membrane fusion events, we have examined the role that each of its three domains play in how NSF interacts with the SNAP SNARE: complex. Mutagenesis of the first ATP-binding domain (D2, amino acids 206-477) demonstrates that n ucleotide binding by this domain is required for 20 S particle assembl y, a second mutation, which permits ATP binding but not hydrolysis, yi elds a protein that can form 20 S particle but fails to mediate its di sassembly. Similar mutations of the second ATP-binding domain (D2, ami no acids 478-744) result in trimeric molecules that behave like wild t ype NSF. Domain rearrangement mutants were used to further probe the f unctional role of each domain. The amino-terminal domain (N, amino aci ds 1-205) is absolutely required for binding of NSF to the SNAP-SNARE complex, because the truncated mutant, D1D2, is unable to form 20 S pa rticle. When tested as an isolated recombinant protein, the N domain i s not sufficient for binding to the SNAP SNARE complex, but when adjac ent to the D1 domain or in a trimeric molecule, the N domain does medi ate binding to the SNAP-SNARE complex. Monomeric N-D1 and trimeric N-D 2 could both participate in particle formation. Only the N-D1 mutant w as able to facilitate MgATP-dependent release from the SNAP-SNARE comp lex. These data demonstrate that NSF binding to the SNAP-SNARE complex is mediated by the N domain and that both ATP binding and hydrolysis by the D1 domain are essential for 20 S particle dynamics. The intramo lecular interactions outlined suggest a mechanism by which NSF may use ATP hydrolysis to facilitate the vesicle fusion process.