Microfilaments are major cytoskeletal elements of all eucaryotic cells. A large number of proteins that associate with actin in higher cells have been identified and characterized, but their in vivo role is frequently poorly understood. The ubiquitious nature of microfilaments, and their altered state in oncogenically transformed cells stresses the importance of studying their biological functions. The time has come to analyze all the microfilament-associated proteins in one cell type and pinpoint the biological role of each protein. For such a project it, is desirable to use a combined biochemical, genetic and cell biological approach. We have therefore chosen the budding yeast, Saccharomyces cerevisiae, as our experimental organism as it is amenable to detailed genetic analysis and contains a single essential actin gene. We have begun to isolate microfilament- associated proteins from this organism, including actin, a trophomysin-like protein, and a putative Ca2+-regulated F-actin severing protein. We propose to characterize these proteins in detail and compare them with their higher cell counterparts to determine both their properties and how useful yeast will be as a model system. We also propose to search for and characterize other microfilament associated proteins in yeast extracts. We then propose to prepare antibodies to each of these proteins to localize them in yeast cells and to isolate their respective genes from a DNA library in the Lambda gt11 expression vector. When the genes have been isolated gene disruption experiments will be used to determine whether the proteins perform essential functions. If these disruptions in haploids result in cell death, we will embark on a project to isolate temperature-sensitive mutations in each of the genes. We will then explore the cell biological consequences of the temperature-sensitive mutations in these cells at the restrictive temperature. This will lead into a future genetic analysis of extragenic suppressors to identify interacting microfilament-associated proteins. By this work, we expect to determine, both biochemically and genetically, whether yeast is a good model system for studying the functional organization of microfilaments. If so, it should set the stage for the thorough analysis of these important cytoskeletal elements.
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