N-Ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment proteins (SNAP) mediate dissociation of GS28-syntaxin 5 Golgi SNAP receptors (SNARE) complex

Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) GS28 and syntaxin 5 can be reciprocally coimmunoprecipitated from Golgi extracts, suggesting that they exist in a protein complex. When Golgi extract is preincubated with soluble NSF attachment proteins (alpha-SNAP) and N-ethylmaleimide-sensitive factor (NSF) under conditions that allow ATP hydrolysis by NSF, GS28 and syntaxin 5 become dissociated. GS28 and syntaxin 5 remain in a protein complex when Golgi extract is preincubated with similar amounts of alpha-SNAP and NSF under conditions that prevent ATP hydrolysis by NSF, suggesting that ATP hydrolysis by NSF is necessary for dissociating the GS28-syntaxin 5 complex. Since preincubation of Golgi extract with either alpha-SNAP or NSF alone has no effect on the GS28-syntaxin 5 complex, a concerted action of alpha-SNAP and NSF therefore mediates the dissociation of the GS28-syntaxin 5 complex. Furthermore, GS28 but not syntaxin 5 is capable of binding to immobilized alpha-SNAP when the GS28-syntaxin 5 complex is dissociated.