Stimulation of NSF ATPase activity by α-SNAP is required for SNARE complex disassembly and exocytosis

Abstract

N-ethylmaleimide-sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH- terminal amino acids resulted in a marked decrease. Both NH2-terminal (1- 160) and COOH-terminal (160-295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1-285) and α-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1-285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1-285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1-285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle- associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α- SNAP stimulation of exocytosis.