Calcium ions regulate a number of processes essential for the growth and development of an individual. Cell division, cellular architecture, and the early stages in development are all regulated by increases in the intracellular concentration of calcium signalled by extracellular messengers. Calmodulin is a ubiquitous calcium-binding protein believed to mediate cellular responses to calcium fluxes. Upon binding Ca2+, calmodulin undergoes conformational transitions that trigger recognition and regulation of target proteins. Potential targets include enzymes of cyclic nucleotide metabolism, the Ca2+- pumping ATPase, cytoskeletal proteins, protein kinases and a protein phosphatase. All of these proteins are regulated by calmodulin in a calcium-dependent manner in vitro, but it has been difficult to study the regulation in vivo. Furthermore, the interactions between calmodulin and its targets are not fully understood at the molecular level. A powerful approach for characterizing calmodulin and its interactions with cellular targets is a combined genetic and biochemical analysis. For this purpose, the yeast Saccharomyces cerevisiae offers considerable experimental advantage because it is amenable to genetic manipulations. Furthermore, calmodulin is essential for the growth of yeast cells. Vertebrate calmodulin can substitute for yeast calmodulin in vivo, therefore an analysis of yeast calmodulin will provide information about vertebrate calmodulin. First, yeast mutants that carry conditionally lethal defects in calmodulin will be isolated. Then, mutations in the genes encoding the target proteins will be isolated by their ability to suppress the conditionally lethal phenotype of the calmodulin mutants or because they confer a dependence on overproduction of calmodulin. (Yeast cells that overproduce calmodulin 80-fold are viable.) Substantial information about the function of calmodulin will be obtained by characterizing the mutants physiologically. The essential structural determinants of calmodulin will be identified by correlating the sequence changes in the mutant proteins with their altered biochemical properties. Special emphasis will be placed on a study of the interactions between mutant calmodulins and mutant and wild type target proteins. In this way, the details of the interactions between calmodulin and target proteins will be elucidated. In the last two years of the proposal, the target proteins will be more thoroughly characterized and a mutational analysis of their function begun.
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