ANALYSIS OF A YEAST SNARE COMPLEX REVEALS REMARKABLE SIMILARITY TO THE NEURONAL SNARE COMPLEX AND A NOVEL FUNCTION FOR THE C-TERMINUS OF THE SNAP-25 HOMOLOG, SEC9

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryo tic cells. The binding properties of the neuronal proteins synaptobrev in 1 (VAMP1), syntaxin 1, SNAP-25, and (s) under bar oluble (N) under bar-ethylmaleimide-(s) under bar ensitive (f) under bar actor (a) unde r bar ttachment (p) under bar rotein (alpha-SNAP), have been extensive ly studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocyt ic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolo g, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neur onal proteins is that individually neither of the target membrane SNAR Es (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synap tobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1 -Sec9 complex for t-SNARE function suggests that regulating the format ion of this complex may be a key step in determining the site of vesic le fusion. In addition, we have used this in vitro assay to examine th e biochemical effects of several mutations in Sec9 that result in pron ounced growth defects in vivo. As expected, a temperature-sensitive mu tation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec 9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 a nd Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects i n vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest tha t the C-terminal domain of Sec9 is specifically required for a novel i nteraction that is required at a step following SNARE assembly.