Abstract
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 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.
Highlights
Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)1 receptors (SNAREs) comprise several families of evolutionarily conserved, membrane-localized receptors for components of the vesicle docking and fusion machinery in eukaryotes
Mutant Analysis of Snc v-SNAREs: Deletion Mutagenesis of Snc1—We have shown that yeast bearing null alleles of SNC1 and SNC2 display conditional-lethal phenotypes that result from a blockage in cellular secretion [3]
Snc proteins localize to secretory vesicles [3], interact with t-SNAREs from the plasma membrane (9, 10, and this study), and are likely to undergo a dynamic cycle of transport to, and retrieval from, the plasma membrane
Summary
JG90, JG92, JG120, JG263, JG402, JG403, JG415, JG506, JG507, JG509, JG510, 59-AACGTATTCGTCGACCATGTCGTC-39 59-CTACATATGGGAGCTCCCTAT-39 59-AGCGACACAGAGCTCCATTTATAGATCCTT-39 59-GAACTTCTATGAGCTCTAGTGAACAG-39 59-GCTCTATCCGAGTCGACTATGGAACGACCCC-39 59-GAATGTACAGTCGACGATGAGGACTGCGG-39 59-GATAACATAAAGTCGACAATGGAAAGAGGTGAAA-39 59-GTACCACATGGCTTTTCTTCTTTCACCTCTTTCTGC-39 59-GCAGAAAGAGGTGAAAGAAGAAAAGCCATGTGGTAC-39 59-TCTAAGTCAAGGACTGCGGCAGAAAGAGGTGAAGA-39 59-TCTTTCACCTCTTTCTGCCGCAGTCCTTGACTTAGA-39. To address whether specific regions within the synaptobrevin/VAMPs confer the SNARE-SNARE interactions that lead to exocytosis, we have used the yeast system to perform a structure/function analysis of the Snc v-SNAREs. By using genetic and biochemical assays, we demonstrate that the ability of Snc proteins to confer exocytic function in vivo corresponds to their ability to interact genetically with components of the putative fusion complex (Sec, Sec, and Sso). We demonstrate that the H1 and H2 segments together mediate the exocytic functions of these v-SNAREs. In addition, there is a distinct requirement for the membrane-spanning domain but no requirement for the intravesicular and variable regions. There is a distinct requirement for the membrane-spanning domain but no requirement for the intravesicular and variable regions The latter suggests that this region is unlikely to act in a fusogenic capacity, as proposed for the amino terminus of the hemagglutinin antigen (HA), a viral fusion protein bearing similar structural motifs (reviewed in Ref. 21)
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