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.

钙是几乎所有细胞信号转导的第二信使。 真核细胞。虽然对控制的机制知道得很多 并对钙信号做出反应，这一过程的许多重要组成部分 没有被确认身份。例如，钙离子信号转导对于 人类T细胞对抗原的正常反应，但关键的钙离子 质膜中的通道尚未确定，这些事件 钙调神经磷酸酶下游，一种依赖于钙离子的蛋白磷酸酶，没有 已经被完全定性了。我们的初步结果显示， 萌芽酵母酿酒酵母也会产生钙信号，并可能 要做到这一点，请使用保守的机制。来自我们的最重要的结论是 研究表明，钙离子在细胞内的信号传导机制尚属首次。 酵母菌可以用遗传方法进行解剖。长期目标 这项建议的主要目的是利用 系统，并制作了详细的钙信号分子模型和 钙动态平衡的相关过程。 我们的初步工作发现了两种新的酶，它们有助于控制 胞浆Ca~(2+)是由PMC1编码的一种高亲和力的Ca~(2+)-ATPase，是一种低亲和力的细胞内钙离子。 由VCX1编码的亲和H+/钙离子交换器。基因研究表明 这些钙离子转运体不仅控制钙调神经磷酸酶的激活 钙调蛋白本身也受钙调神经磷酸酶的反馈调节。这个 钙调神经磷酸酶似乎直接或间接地灭活了交换器 而钙调神经磷酸酶明显诱导PMCI基因表达。 这项提案旨在定义钙调神经磷酸酶如何实现这些特定的 效果。我们将克隆并表征下游的调控因素 将它们组织成一个功能序列，确定如何 这些因素会影响靶标以及它们对钙调神经磷酸酶的反应。这个 从这些研究中获得的结果将与 来自哺乳动物细胞内钙信号研究的信息。 我们还提出了新的证据，表明酵母中的钙信号是 生理上很重要。我们观察到了两个新的环境条件 它通过激发可能的钙信号来激活钙调神经磷酸酶。钙离子 这些发现预测的渠道和监管因素目前 未知。因此，我们提出了类似于电容性钙离子进入的观点。 描述了哺乳动物细胞。我们将进行基因筛查， 揭示观察到的钙离子内流增加所需的因素 对细胞内钙池耗尽的反应。要实现这些目标 目标我们将使用专门设计的遗传、分子、细胞 生物、生理和生化技术。