CONSERVED ALPHA-HELICAL SEGMENTS ON YEAST HOMOLOGS OF THE SYNAPTOBREVIN VAMP FAMILY OF V-SNARES MEDIATE EXOCYTIC FUNCTION/

We are studying yeast homologs of the synaptobrevin/VAMP family of ves icle-associated membrane proteins, which act as vesicular compartment- soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) in cells having a capacity for stimulus-coupled secretion, as well as in other cell types. The yeast homologs, Snc1 and Snc2, lo calize to secretory vesicles and are required for normal bulk secretio n in Saccharomyces cerevisiae. Here we have used Snc deletion mutants and chimeric Snc-VAMP proteins to demonstrate that these v-SNAREs can be dissected into regions that are either indispensable or dispensable for exocytic function in vivo. We have found that a region encompassi ng two predicted amphipathic alpha-helices (helix 1 and helix 2) (resi dues 32-85), which are thought to form coiled-coil structures, is esse ntial for conferring exocytosis in yeast. Deletions in either the heli x 1 or helix 2 segments result in a complete loss in the ability of th e protein to confer secretion competence to snc cells and to interact genetically with components of the proposed fusion complex: the Sec9 a nd Sso2 t-SNAREs and the Sec17 alpha-SNAP homolog. In contrast, deleti ons in either the variable (residues 2-27) or putative intravesicular (residues 115-117) regions have no deleterious effect upon v-SNARE fun ction. This makes it unlikely that sequences in either the amino or ca rboxyl terminus act in an exocytic capacity. Along with additional stu dies utilizing chimeric Snc-VAMP proteins, we suggest that although th e Snc and synaptobrevin/VAMP proteins have evolved to mediate vastly d ifferent exocytic programs, their structural requirements and actions have remained remarkably well-conserved in evolution.