DOMAINS OF ALPHA-SNAP REQUIRED FOR THE STIMULATION OF EXOCYTOSIS AND FOR N-ETHYLMALEMIDE-SENSITIVE FUSION PROTEIN (NSF) BINDING AND ACTIVATION

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sen sitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one oi more of these membrane prote ins that are essential for regulated exocytosis. Functional evidence f or a role of alpha-SNAP in exocytosis in adrenal chromaffin cells come s from the ability of this protein to stimulate Ca2+-dependent exocyto sis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the abi lity of alpha-SNAP to interact with NSF, to define essential domains i nvolved in protein-protein interactions in exocytosis. Deletion of ext reme N- or C-terminal regions of alpha-SNAP produced proteins unable t o bind to syntaxin or to stimulate exocytosis, suggesting that these d omains participate in essential interactions. Deletion of C-terminal r esidues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to st imulate the ATPase activity of NSF. These results suggest that the C-t erminus, but not the N-terminus, of alpha-SNAP is crucial for interact ions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntax in and NSF would place the latter two proteins in proximity in a terna ry complex whereupon the energy derived from ATP hydrolysis by NSF cou ld induce a conformational change in syntaxin required for exocytosis to proceed.