We are studying yeast homologs of the synaptobrevin/VAMP family of vesicle-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, localize to secretory vesicles and ...

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are required for normal bulk secretion 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 encompassing two predicted amphipathic alpha-helices (helix 1 and helix 2) (residues 32-85), which are thought to form coiled-coil structures, is essential for conferring exocytosis in yeast. Deletions in either the helix 1 or helix 2 segments result in a complete loss in the ability of the protein to confer secretion competence to snc cells and to interact genetically with components of the proposed fusion complex: the Sec9 and Sso2 t-SNAREs and the Sec17 alpha-SNAP homolog. In contrast, deletions in either the variable (residues 2-27) or putative intravesicular (residues 115-117) regions have no deleterious effect upon v-SNARE function. This makes it unlikely that sequences in either the amino or carboxyl terminus act in an exocytic capacity. Along with additional studies utilizing chimeric Snc-VAMP proteins, we suggest that although the Snc and synaptobrevin/VAMP proteins have evolved to mediate vastly different exocytic programs, their structural requirements and actions have remained remarkably well-conserved in evolution.

Date: Jun. 27, 1997

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