Biochemical analysis of the Saccharomyces cerevisiae SEC18 gene product: Implications for the molecular mechanism of membrane fusion

The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide- sensitive fusion protein), and both proteins encode cytoplasmic ATPases whi ch are essential for membrane traffic in yeast and mammalian cells, respect ively. A wealth of biochemical analysis has led to the description of a mod el for the action of NSF; through its interaction with SNAPs (soluble NSF a ttachment proteins), NSF can associate with SNAP receptors (SNAREs) on intr acellular membranes, forming 20S complexes. SNAPs then stimulate the intrin sic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is b ased almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membra ne fusion is conserved in all eukaryotic cells. If so, the crucial biochemi cal properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here tha t Sec18p can specifically interact not only with Sec17p but also with its m ammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-S NAP stimulation of NSF, although differences in temperature and N-ethylmale imide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNA P to stimulate regulated exocytosis in mammalian cells. We conclude that th e mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved fr om yeast to mammals.