In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6‐Sec8‐Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co‐immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6‐Sec8‐Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.
The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.