J. Cell Biol., Volume 139, Number 4, November 17, 1997 875-883

Stimulation of NSF ATPase Activity by -SNAP Is Required for SNARE Complex Disassembly and Exocytosis

Richard J.O. Barnard, Alan Morgan, and Robert D. Burgoyne

The Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK

N-ethylmaleimide-sensitive fusion protein (NSF) and -SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH₂- and COOH-terminal truncation mutants of -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 -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 -SNAP were able to bind to NSF, suggesting that -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 -SNAP. Mutation of leucine 294 to alanine (-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. -SNAP (1-285) and -SNAP (L294A) were unable to stimulate Ca²⁺-dependent exocytosis in permeabilized chromaffin cells. In addition, -SNAP (1-285), and -SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous -SNAP. -SNAP, -SNAP (1-285), and -SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, -SNAP (1-285) and -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 -SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the -SNAP stimulation of exocytosis.

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