Each Domain of the N-Ethylmaleimide-sensitive Fusion Protein Contributes to Its Transport Activity

Abstract

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. 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.