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 involved in numerous intracellular transport events. In an effort to understand 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 (D1, amino acids 206-477) demonstrates that nucleotide binding by this domain is required for 20 S particle assembly. A second mutation, which permits ATP binding but not hydrolysis, yields a protein that can form 20 S particle but fails to mediate its disassembly. Similar mutations of the second ATP-binding domain (D2, amino acids 478-744) result in trimeric molecules that behave like wild type NSF. Domain rearrangement mutants were used to further probe the functional role of each domain. The amino-terminal domain (N, amino acids 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 particle. When tested as an isolated recombinant protein, the N domain is not sufficient for binding to the SNAP.SNARE complex, but when adjacent to the D1 domain or in a trimeric molecule, the N domain does mediate binding to the SNAP.SNARE complex. Monomeric N-D1 and trimeric N-D2 could both participate in particle formation. Only the N-D1 mutant was able to facilitate MgATP-dependent release from the SNAP.SNARE complex. 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 intramolecular interactions outlined suggest a mechanism by which NSF may use ATP hydrolysis to facilitate the vesicle fusion process.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D011817:Rabbits; D013367:Submitochondrial Particles / Q000737:chemistry; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins