SNAP PREVENTS MG2-ATP-INDUCED RELEASE OF N-ETHYLMALEIMIDE-SENSITIVE FACTOR FROM THE GOLGI-APPARATUS IN DIGITONIN-PERMEABILIZED PC12 CELLS()

The N-ethylmaleimide-sensitive factor (NSF), which is involved in the multisteps of protein transport, is released from Golgi membranes on i n vitro incubation with Mg2+-ATP. However, several lines of evidence s uggest that NSF is associated with membranes in spite of the presence of Mg2+ and ATP in vivo. We have used digitonin-permeabilized PC12 cel ls to investigate the mechanism underlying the association of NSF with membranes, In PC12 cells, immunoreactivity for NSF was observed in th e nuclear membranes, the Gels ap paratus, and neuronal growth cones, w here synaptic vesicles are concentrated, NSF associated with the Golgi apparatus was released on incubation with Mg2+-ATP, whereas NSF in th e nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg2+-ATP-induced r elease of NSF from the Golgi apparatus. Chromatographic analyses revea led that the factor(s) that prevents NSF release from the Golgi appara tus was eluted at the same position as the soluble NSF attachment prot eins (SNAPs). Purified His(6)-tagged alpha-SNAP exhibited such activit y. His(6)-tagged alpha-SNAP also prevented the Mg2+-ATP-induced releas e of NSF from isolated Golgi membranes.