Intracellular membrane fusion is crucial for the secretory and endocytotic pathways. Recently, great advances have been made in identifying proteins which have been implicated in playing crucial roles in these processes. Among these proteins is N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) and a whole family of proteins with which it interacts. We have used 2 different preparations in order to investigate the involvement of NSF in intracellular fusion events. These are the Ca2+-stimulated exocytosis of sea urchin egg cortical granules and the GTP-triggered fusion of rat liver endoplasmic reticulum microsomes. Cortical granule exocytosis is an ATP- and cytosol-independent Ca2+-stimulated membrane fusion event which is inhibited by NEM. We have established that cytosol is unable to restore fusion to the isolated exocytotic machinery inactivated by NEM. We have found using immunoblot analysis that sea urchin eggs contain NSF in their cytosol but that this protein is absent from an in vitro preparation which undergoes fusion upon the provision of Ca2+. However, a stable and specific interaction between NSF and the isolated exocytotic machinery can occur under the appropriate conditions. These results suggest that the role of NSF might be in the establishment of a fusion-competent arrangement rather than in membrane fusion per se. Similarly, we found that the sensitivity of the homotypic fusion of endoplasmic reticulum membranes to NEM appeared to have characteristics distinct from those observed in reconstituted membrane transport and associated with NSF. Now, we have shown that additional purification and extraction of endoplasmic reticulum membranes with high salt that increases the GTP-dependent fusion activity of the membranes results in complete removal of the small amounts of NSF detectable in the """"""""crude"""""""" membrane preparation. In addition, we found that anti-NSF monoclonal antibodies which are known to functionally inhibit NSF-dependent fusion had no effect on the fusion of endoplasmic reticulum membranes. These data corroborate our previous results and support the conclusion that the NEM-sensitive component required for GTP-dependent fusion of endoplasmic reticulum membranes is distinct from NSF. This suggests a novel, NSF-independent mechanism of fusion.
