THE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN AND ALPHA-SNAP INDUCE ACONFORMATIONAL CHANGE IN SYNTAXIN

The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of thi s complex by NSF is thought to be a critical step in the molecular eve nts which lead to vesicle fusion with the plasma membrane. Here we hav e studied the interaction of alpha-SNAP and NSF with individual compon ents of this complex and have identified syntaxin as a primary substra te for NSF/alpha-SNAP. We find that alpha-SNAP binds directly to synta xin 1A as well as weakly to SNAP-25, while it does not bind to synapto brevin II. NSF binds to syntaxin through alpha-SNAP and in the presenc e of ATP catalyzes a conformational rearrangement which abolishes bind ing of itself and alpha-SNAP. This reaction leads to the previously de scribed disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced, alpha-SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of alpha-SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on a n interaction between carboxyl- and amino-terminal do mains of syntaxi n.