Relatively little is known about the function of myosin in non-muscle systems. The goal of this proposal is to elucidate the physiological role of myosin and its regulation in a non-muscle system. We propose to conduct these studies in the yeast S. cerevisiae. Yeast represents an advantageous system in which to conduct these studies because it can be easily cultured and manipulated, the methodologies for traditional and molecular genetic manipulations have been developed and genetic mutants in other structural protein genes are readily available. In this study, biochemical, traditional genetic and molecular genetic approaches will be used to identify and characterize the functional regions of yeast myosin. The role of myosin in the assembly of intracellular actin-myosin fibers and the function of such assemblies in cellular processes will be investigated. In addition to our structural studies, we will analyze the regulation of myosin throughout the cell cycle. Training of students at graduate and undergraduate level is stressed.
The specific aims are: 1) to test the hypothesis that myosin is an essential component of the yeast cytoskeleton, by using immunochemical methods to study the localization of actomyosin assemblies in myosin and actin mutants; 2) to test the hypothesis that myosin is regulated during the yeast cell cycle, by quantitatively analyzing myosin mRNA and protein in synchronized cultures; 3) to test the hypothesis that regulation by phosphorylation at the carboxy terminus of yeast myosin is important for myosin function, by SDS-PAGE analysis of myosin labelled with [32P]P1 in vivo, in vitro mutagenesis, cell transformation, and functional analysis of mutants in vivo and; 4) to train students in molecular biology techniques with the expectation that graduate degrees will be pursued.
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