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 which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.
Steel
G J
GJ
The Physiological Laboratory, University of Liverpool, United Kingdom.
Laude
A J
AJ
Boojawan
A
A
Harvey
D J
DJ
Morgan
A
A
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; C073478:MRP20 protein, S cerevisiae; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide