MOLECULAR ANALYSIS OF CALMODULIN FUNCTION IN CELL GROWTH

钙离子调节许多过程，这些过程对细胞的生长至关重要。 个人的成长和发展。 细胞分裂，细胞 架构，以及开发的早期阶段， 通过增加细胞内 钙通过细胞外信使发出信号。 钙调素是一种普遍存在的钙结合蛋白， 介导细胞对钙流的反应。 在结合Ca 2+后， 钙调素经历构象转变， 识别和调节靶蛋白。 潜在目标 包括环核苷酸代谢酶，Ca 2 +- 泵ATP酶，细胞骨架蛋白，蛋白激酶和 蛋白磷酸酶 所有这些蛋白质都受到 钙调素在体外以钙依赖的方式，但它有 在体内研究其调控机制是困难的。 而且 钙调素与其靶点之间的相互作用并不完全 在分子水平上理解。 一种强有力的方法来表征钙调素及其 与细胞靶点的相互作用是一种组合的遗传和 生化分析 为此，酵母菌 酿酒厂提供了相当大的实验优势，因为它 是可以被基因操控的 此外，钙调素是 对酵母细胞的生长至关重要。 脊椎动物钙调素能 在体内替代酵母钙调素，因此， 酵母钙调素将提供有关脊椎动物 钙调素 首先，携带条件致死缺陷的酵母突变体， 将分离钙调蛋白。 然后，基因突变 编码靶蛋白的蛋白质将通过它们的能力被分离， 抑制钙调蛋白的条件致死表型 突变体或因为它们赋予了对过度生产的依赖性 钙调素 （过量产生钙调素80倍的酵母细胞 是可行的）。 关于功能的大量信息 钙调蛋白将通过表征突变体来获得 生理上的。 钙调素的基本结构决定因素是 通过关联突变体中的序列变化来鉴定 改变了生物化学性质的蛋白质。 特别 重点将放在研究之间的相互作用， 突变型钙调蛋白和突变型和野生型靶蛋白。 在 这样，钙调蛋白和 将阐明靶蛋白。 在过去的两年里， 建议，目标蛋白质将更彻底 并开始对其功能进行突变分析。

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.