CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE

钙作为第二信使的信号转导几乎所有 真核细胞 虽然我们对控制这种疾病的机制已经有了很多了解， 并对Ca 2+信号作出反应，这一过程的许多重要组成部分 未被确认。 例如，Ca 2+信号传导是必需的， 人T细胞对抗原的正常反应，但关键的Ca 2 + 细胞膜中的通道尚未确定， 钙调神经磷酸酶（一种钙离子依赖性蛋白磷酸酶）的下游， 被完全定性。 我们的初步结果显示， 芽殖酵母酿酒酵母也产生Ca 2+信号， 使用保守的机制。 我们得出的最重要的结论是 研究表明，第一次，Ca 2+信号转导的机制， 酵母可以用遗传学方法进行解剖。 长期目标 这一建议的目的是利用实验的优势， 系统，并产生一个详细的分子模型的Ca 2+信号和 Ca 2+稳态的相关过程。 我们的初步工作揭示了两种新的酶， 胞浆Ca ~（2+）、由PMC 1编码的高亲和力Ca ~（2+）ATP酶和低亲和力Ca ~（2+）ATP酶。 由VCX 1编码的亲和H+/Ca 2+交换剂。 遗传学研究表明， 这些Ca 2+转运蛋白不仅通过 钙调素，但本身是由钙调神经磷酸酶反馈调节。 的 钙调神经磷酸酶似乎直接或间接地使交换器失活 而PMCI基因的表达似乎是由钙调神经磷酸酶显著诱导的。 该提案旨在定义钙调神经磷酸酶如何完成这些特定的 方面的影响. 我们将克隆和表征下游的调节因子 将它们组织成一个功能序列， 这些因素影响靶点以及它们对钙调磷酸酶的反应。 的 从这些研究中获得的结果将是有趣的， 这些信息来自哺乳动物细胞中Ca 2+信号的研究。 我们还提出了新的证据，表明酵母中的Ca 2+信号是 生理重要性。 我们观察到两种新的环境条件 其通过引起推定的Ca 2+信号来激活钙调磷酸酶。 的ca 2 + 这些发现预测的通道和调节因素目前 未知 因此，我们建议类似的容量Ca 2+进入， 描述了哺乳动物细胞。 我们将进行基因筛查， 揭示了增加Ca 2+流入所需的因素， 对细胞内Ca 2+池耗竭的反应。 完成这些 我们将使用专门设计的基因，分子，细胞 生物学、生理学和生物化学技术。

Calcium serves as a second messenger for signal transduction in nearly all eukaryotic cells. While much is known about the mechanisms which control and respond to Ca2+ signals, many important components of this process have not been identified. For example, Ca2+ signaling is necessary for the normal response of human T-cells to antigens, yet the critical Ca2+ channels in the plasma membrane have not been identified and the events downstream of calcineurin, a Ca2+-dependent protein phosphatase, have not been completely characterized. Our preliminary results reveal that the budding yeast Saccharomyces cerevisiae also produces Ca2+ signals and may do so using conserved mechanisms. The most significant conclusion from our studies is that, for the first time, the mechanism of Ca2+ signaling in yeast can be dissected using genetic approaches. The long-term objective of this proposal is to take advantage of the experimental strengths of the system and produce a detailed molecular model of Ca2+ signaling and the related process of Ca2+ homeostasis. Our preliminary work has revealed two new enzymes that help control cytosolic Ca2+, a high-affinity Ca2+ ATPase encoded by PMC1 and a low- affinity H+/Ca2+ exchanger encoded by VCX1. Genetic studies show that these Ca2+ transporters not only control the activation of calcineurin by calmodulin, but are themselves feedback regulated by calcineurin. The exchanger appears to be directly or indirectly inactivated by calcineurin whereas PMCI gene expression appears to be markedly induced by calcineurin. This proposal aims to define how calcineurin accomplishes these specific effects. We will clone and characterize the regulatory factors downstream of calcineurin, organize them into a functional sequence, determine how these factors affect the targets and how they respond to calcineurin. The results obtained from these studies will be interesting to compare with the information emerging from studies of Ca2+ signaling in mammalian cells. We also present new evidence that Ca2+ signaling in yeast is physiologically important. We observed two new environmental conditions which activate calcineurin by evoking putative Ca2+ signals. The Ca2+ channels and regulatory factors predicted by these findings are currently unknown. Accordingly, we propose resembling capacitative Ca2+ entry in mammalian cells is described. We will perform genetic screens that should reveal the factors required for the increased Ca2+ influx observed as a response to depletion of intracellular Ca2+ pools. To accomplish these goals we will use specifically designed genetic, molecular, cell biological, physiological, and biochemical techniques.