STIMULATION OF NSF ATPASE ACTIVITY BY ALPHA-SNAP IS REQUIRED FOR SNARE COMPLEX DISASSEMBLY AND EXOCYTOSIS

N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play ke y roles in vesicular traffic through the secretory pathway. In this st udy, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assa yed for ability to bind NSF and stimulate its ATPase activity. Deletio n of up to 160 NH2-terminal amino acids had little effect on the abili ty of alpha-SNAP to stimulate the ATPase activity of NSF. However, del etion of as few as 10 COOH-terminal amino acids resulted in a marked d ecrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragmen ts of alpha-SNAP were able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequen ce alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to s timulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were una ble to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285), and alpha-SNAP (L294A) were a ble to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) were all able t o become incorporated into a 20S complex and recruit NSF. In the prese nce of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associate d membrane protein dissociation to any greater level than seen in cont rol incubations. These findings imply that alpha-SNAP stimulation of N SF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis.