MOLECULAR ANALYSIS OF THE YEAST ACTIN CYTOSKELETON

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The actin cytoskeleton has been implicated in a large variety of motile functions, as well as in the structural and mechanical properties of the cytoplasmic matrix. Except in muscle cells, however, very little is known about its composition, roles, or mechanism of action. The proposed research is designed to determine at the molecular level how the actin cytoskeleton functions in the yeast Saccharomyces cerevisiae. Yeast can be readily manipulated genetically, and should allow rapid and detailed analysis of these complex processes. Because actin is highly conserved from yeast to man, what is learned from these studies is likely to apply to higher cells, where defective actin cytoskeleton function is implicated in pathological conditions such as muscular dystrophy and neoplastic transformation. The following specific questions will be addressed: 1. How does SAC6p, an actin-binding protein, function in the cytoskeleton? Conditional-lethal mutations in the SAC6 gene will be produced by in vitro mutagenesis. Biochemical and physiological analyses of a full spectrum of mutants will then allow activities observed in vitro to be correlated with functions seen in vivo. 2. What residues or domains are important for function of SAC6p and its higher-cell homologs? Fine-structure mapping of mutations will be used to identify regions of the gene associated with various C6p activities. Specific hypotheses concerning sequences such as the putative actin-binding domain (a highly conserved 25 amino acid sequence found in a number of actin-binding proteins) will be tested by site-specific mutagenesis. Information from these studies will be used in an analysis of human plastin and chicken fimbrin, which are 40% identical to SAC6p. 3. What other proteins are involved in the function of the actin cytoskeleton? The diversity of actin cytoskeletal function must be mediated by non-actin proteins that regulate actin assembly, establish interactions between the cytoskeleton and other cell structures, and generate the force for intracellular motility. A variety of genetic methods will be used to identify additional components of the actin cytoskeleton, and proteins that interact with it.