SYNAPTIC CORE COMPLEX OF SYNAPTOBREVIN, SYNTAXIN, AND SNAP25 FORMS HIGH-AFFINITY ALPHA-SNAP FINDING SITE

SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins that bind to specific membrane receptors and mediate the membrane binding o f NSF (N-ethylmaleimide-sensitive factor), a protein that is required for membrane fusion reactions. Three synaptic proteins in brain (SNAP2 5 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were identified as SNAP r eceptors by affinity chromatography on immobilized alpha-SNAP complexe d to NSF (Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Broma ge, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature 362 , 318-324). However, the nature of the alpha-SNAP binding site is uncl ear. We now show that alpha-SNAP binds tightly to the complex of synta xin with synaptobrevin. SNAP25 is not required for tight binding of al pha-SNAP to this complex but stabilizes the syntaxin synaptobrevin com plex by forming a trimeric core complex with it. alpha-SNAP does not b ind to synaptobrevin individually and binds only weakly to syntaxin an d SNAP25 in the absence of synaptobrevin. These data suggest that the complex of the vesicular protein synaptobrevin with the plasma membran e protein syntaxin is required for physiological alpha-SNAP binding. T hus, alpha-SNAP probably functions in a late step of the membrane fusi on reaction after the formation of the synaptobrevin-syntaxin-SNAP25 c ore complex.