MOLECULAR ANALYSIS OF CALMODULIN FUNCTION IN CELL GROWTH

钙离子调节许多过程，这些过程对于 个体的成长和发展。细胞分裂，细胞分裂 架构，以及开发的早期阶段都是 通过细胞内浓度的增加进行调节 钙离子由细胞外信使发出信号。 钙调蛋白是一种普遍存在的钙结合蛋白，据信 调节细胞对钙离子通量的反应。当结合钙离子时， 钙调蛋白经历构象转换触发 靶蛋白的识别和调控。潜在目标 包括环核苷酸代谢酶，钙离子- 泵ATPase、细胞骨架蛋白、蛋白激酶和a 蛋白质磷酸酶。所有这些蛋白质都受 钙调素在体外以钙依赖的方式，但它 在体内的调控研究一直比较困难。此外， 钙调蛋白与其靶点之间的相互作用还不完全 在分子水平上被理解。 一种表征钙调蛋白的有效方法及其应用 与细胞靶点的相互作用是一种遗传和 生化分析。为此，酵母菌 酿酒提供了相当大的实验优势，因为它 是可以接受基因操纵的。此外，钙调蛋白是 酵母细胞生长所必需的。脊椎动物钙调蛋白可以 在体内替代酵母钙调素，因此分析 酵母钙调素将提供有关脊椎动物的信息 钙调素。 首先，携带有条件致命性缺陷的酵母突变体 钙调素将被分离出来。然后，基因的突变 编码目标蛋白的能力将被分离出来 抑制钙调素的条件性致死表型 突变者或因为他们对生产过剩的依赖 钙调素。(过量产生钙调蛋白的酵母细胞是原来的80倍 是可行的。)关于的功能的大量信息 将通过鉴定突变体获得钙调蛋白 生理上的。 钙调蛋白的基本结构决定因素是 通过关联突变体中的序列变化进行鉴定 具有改变的生化特性的蛋白质。特价 重点将放在研究相互作用上。 突变型钙调蛋白和突变型和野生型靶蛋白。在……里面 这样，钙调素和钙调素之间相互作用的细节 靶蛋白将被阐明。在过去的两年里 建议，靶蛋白将更加彻底 并开始对它们的功能进行突变分析。

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.