CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE

酿酒酵母中的钙信号传导和运输

• 批准号: 2900845
• 负责人: KYLE W CUNNINGHAM
• 金额: $ 25.59万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900845
• 关键词: Saccharomyces cerevisiae; biological signal transduction; calcineurin; calcium channel; calcium flux; calcium indicator; calcium transporting ATPase; gene complementation; gene expression; homeostasis; membrane transport proteins; molecular cloning; phenotype; polymerase chain reaction; second messengers; transcription factor

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.

钙是几乎所有的信号转导的第二信使 真核细胞。 虽然对控制的机制知之甚少 并响应CA2+信号，此过程的许多重要组成部分 未被确定。 例如，Ca2+信号传导是必需的 人类T细胞对抗原的正常反应，但是关键的Ca2+ 尚未确定质膜中的通道和事件 钙调神经酶的下游是一种Ca2+依赖性蛋白质磷酸酶，尚未 被完全表征。 我们的初步结果表明 酿酒酵母发芽的酵母糖酵母也会产生CA2+信号，可能 使用保守的机制执行此操作。 我们最重要的结论 研究是，第一次是Ca2+信号传导的机理 可以使用遗传方法解剖酵母。 长期目标 该提议是利用 系统并产生Ca2+信号传导的详细分子模型和 CA2+稳态的相关过程。 我们的初步工作揭示了两种有助于控制的新酶 胞质Ca2+，一种由PMC1编码的高亲和力Ca2+ ATPase 亲和力H+/Ca2+由VCX1编码的交换器。 遗传研究表明 这些CA2+转运蛋白不仅控制着钙调神经酶的激活 钙调蛋白，但本身是由钙调蛋白调节的反馈。 这 交换器似乎直接或间接被钙调蛋白灭活 而PMCI基因表达似乎是由钙调蛋白明显诱导的。 该建议旨在定义钙调神经酶如何完成这些特定 效果。 我们将克隆并表征下游的调节因子 钙调蛋白酶，将它们组织成功能序列，确定如何 这些因素会影响靶标及其对钙调神经酶的反应。 这 从这些研究获得的结果将与 从哺乳动物细胞中Ca2+信号传导的研究中出现的信息。 我们还提供了新的证据，表明酵母中的Ca2+信号传导为 生理上很重要。 我们观察到了两个新的环境条件 通过引起推定的Ca2+信号来激活钙调神经素。 Ca2+ 这些发现预测的渠道和监管因素目前是 未知。 因此，我们提出类似于电容的Ca2+进入 描述了哺乳动物细胞。 我们将执行应 揭示了观察到的Ca2+涌入所需的因素 对细胞内Ca2+池耗竭的反应。 完成这些 目标我们将使用专门设计的遗传，分子，细胞 生物学，生理和生化技术。