We are interested in the biology of the actin cytoskeleton, in particular how actin-binding proteins regulate its assembly and organization. We propose to study these questions in the yeast Saccharomyces cerevisae because this system has good genetics, molecular biology, biochemistry and cell biology. To begin, we have identified the yeast homologue of capping protein, a ubiquitous actin-binding protein that regulates actin assembly, and propose to study its role in the actin cytoskeleton and identify interacting genes. The protein, which has alpha and beta subunits, was purified. The corresponding genes, CAP1 and CAP2, were cloned, sequenced and deleted. Antibody localization shows capping protein in association with cortical patches of actin, but not cables or the cytokinesis ring. The null mutant is viable alone but lethal in combination with null mutations of fimbrin, SAC6. To analyze the role of capping protein in vivo, we will examine the phenotype of cells that lack capping protein or have an excess of capping protein using gene deletion and overexpression strains. We will focus on aspects of cell physiology in which the actin cytoskeleton may be involved, including actin distribution, bud site selection, cell morphology, growth rate, mating morphogenesis and secretion. We will observe the development of the null phenotype over time in conditional mutants. To understand the function of capping protein in vivo, we will determine whether cell phenotype is affected by point mutations that alter functions of capping protein that have been defined in vitro, specifically capping protein's ability to regulate actin polymerization and its inhibition by anionic phospholipids. In addition, identification of mutants with an abnormal phenotype but normal in vitro activities will indicate that capping protein has functions or interactions other than these two, such as binding to the plasma membrane. The phenotype of the transformants with mutant genes on plasmids will be analyzed, and capping protein will be partially purified and analyzed for in vitro activity, thus correlating phenotype with in vitro activity. We will identify genes for proteins that bind to capping protein and genes for proteins that have similar or related functions using several approaches. 1. Suppression analysis of the capping protein null mutants. The null strain will be mutagenized or transformed with an overexpression plasmid library and selected for growth at restrictive conditions. 2. Suppression analysis of the point mutants of capping protein, generated above, to identify molecules other than actin and anionic phospholipids to which capping protein binds and to analyze the significance of anionic phospholipid inhibition. 3. Because actin is essential for viability and capping protein is not, mutations in other actin regulatory proteins might be lethal in combination with mutations in capping protein. We will screen for such genes by mutagenizing a capping protein null strain with capping protein on a counter-selectable plasmid.
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