Synaptic Core Complex of Synaptobrevin, Syntaxin, and SNAP25 Forms High Affinity -SNAP Binding Site

SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins that bind to specific membrane receptors and mediate the membrane binding of NSF (N-ethylmaleimide-sensitive factor), a protein that is required for membrane fusion reactions. Three synaptic proteins in brain (SNAP25 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were identified as SNAP receptors by affinity chromatography on immobilized α-SNAP complexed to NSF (Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature 362, 318-324). However, the nature of the α-SNAP binding site is unclear. We now show that α-SNAP binds tightly to the complex of syntaxin with synaptobrevin. SNAP25 is not required for tight binding of α-SNAP to this complex but stabilizes the syntaxin-synaptobrevin complex by forming a trimeric core complex with it. α-SNAP does not bind to synaptobrevin individually and binds only weakly to syntaxin and SNAP25 in the absence of synaptobrevin. These data suggest that the complex of the vesicular protein synaptobrevin with the plasma membrane protein syntaxin is required for physiological α-SNAP binding. Thus, α-SNAP probably functions in a late step of the membrane fusion reaction after the formation of the synaptobrevin-syntaxin-SNAP25 core complex. SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins that bind to specific membrane receptors and mediate the membrane binding of NSF (N-ethylmaleimide-sensitive factor), a protein that is required for membrane fusion reactions. Three synaptic proteins in brain (SNAP25 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were identified as SNAP receptors by affinity chromatography on immobilized α-SNAP complexed to NSF (Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature 362, 318-324). However, the nature of the α-SNAP binding site is unclear. We now show that α-SNAP binds tightly to the complex of syntaxin with synaptobrevin. SNAP25 is not required for tight binding of α-SNAP to this complex but stabilizes the syntaxin-synaptobrevin complex by forming a trimeric core complex with it. α-SNAP does not bind to synaptobrevin individually and binds only weakly to syntaxin and SNAP25 in the absence of synaptobrevin. These data suggest that the complex of the vesicular protein synaptobrevin with the plasma membrane protein syntaxin is required for physiological α-SNAP binding. Thus, α-SNAP probably functions in a late step of the membrane fusion reaction after the formation of the synaptobrevin-syntaxin-SNAP25 core complex.