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

doi:10.1083/jcb.139.4.875

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© The Rockefeller University Press, 0021-9525/1997//875 $5.00
The Journal of Cell Biology, Volume 139, Number 4, , 1997 875-883

Article

Stimulation of NSF ATPase Activity by ${alpha}$ -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 ${alpha}$ -SNAPplay key roles in vesicular traffic through the secretory pathway.In this study, NH₂- and COOH-terminal truncation mutants of ${alpha}$ -SNAP were assayed for ability to bind NSF and stimulate itsATPase activity. Deletion of up to 160 NH₂-terminal amino acidshad little effect on the ability of ${alpha}$ -SNAP to stimulate the ATPaseactivity of NSF. However, deletion of as few as 10 COOH-terminalamino acids resulted in a marked decrease. Both NH₂-terminal(1–160) and COOH-terminal (160–295) fragments of ${alpha}$ -SNAP were able to bind to NSF, suggesting that ${alpha}$ -SNAP contains distinct NH₂- and COOH-terminal binding sites for NSF. Sequencealignment of known SNAPs revealed only leucine 294 to be conservedin the final 10 amino acids of ${alpha}$ -SNAP. Mutation of leucine 294to alanine ( ${alpha}$ -SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on theability of this mutant to bind NSF. ${alpha}$ -SNAP (1–285) and ${alpha}$ -SNAP (L294A) were unable to stimulate Ca²⁺-dependent exocytosisin permeabilized chromaffin cells. In addition, ${alpha}$ -SNAP (1–285),and ${alpha}$ -SNAP (L294A) were able to inhibit the stimulation of exocytosisby exogenous ${alpha}$ -SNAP. ${alpha}$ -SNAP, ${alpha}$ -SNAP (1–285), and ${alpha}$ -SNAP (L294A)were all able to become incorporated into a 20S complex andrecruit NSF. In the presence of MgATP, ${alpha}$ -SNAP (1–285) and ${alpha}$ -SNAP (L294A) were unable to fully disassemble the 20S complexand did not allow vesicle-associated membrane protein dissociationto any greater level than seen in control incubations. Thesefindings imply that ${alpha}$ -SNAP stimulation of NSF ATPase activitymay be required for 20S complex disassembly and for the ${alpha}$ -SNAPstimulation of exocytosis.

Abbreviations used in this paper: NBB, NSF binding buffer; NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; SWB, SNAP wash buffer; VAMP, vesicle-associated membrane protein.

Address all correspondence to R.D. Burgoyne, The PhysiologicalLaboratory, University of Liverpool, Crown Street, LiverpoolL69 3BX, UK. Tel.: 44 151 794 5311. Fax: 44 151 794 5337. E-mail:burgoyne{at}liverpool.ac.uk

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