CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE

酿酒酵母中的钙信号传导和运输

• 批准号: 2392245
• 负责人: KYLE W CUNNINGHAM
• 金额: $ 18.77万
• 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/2392245
• 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.

钙作为几乎所有信号转导的第二信使 真核细胞。 虽然人们对控制机制了解很多 并响应 Ca2+ 信号，该过程的许多重要组成部分 未被识别。 例如，Ca2+信号传导对于 人类 T 细胞对抗原的正常反应，但关键的 Ca2+ 质膜中的通道尚未确定，并且事件 钙调神经磷酸酶（一种 Ca2+ 依赖性蛋白磷酸酶）的下游，尚未 已被完全表征。 我们的初步结果表明 芽殖酵母酿酒酵母也产生 Ca2+ 信号，并且可能 使用保守的机制来做到这一点。 我们得出的最重要的结论 研究首次揭示了Ca2+信号传导机制 可以使用遗传方法解剖酵母。 长期目标 该提案的目的是利用 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 胞浆 Ca2+，一种由 PMC1 编码的高亲和力 Ca2+ ATP 酶和一种低亲和力 Ca2+ ATP 酶 VCX1 编码的亲和力 H+/Ca2+ 交换剂。 遗传学研究表明 这些 Ca2+ 转运蛋白不仅通过以下方式控制钙调磷酸酶的激活 钙调蛋白，但其本身是由钙调磷酸酶调节的反馈。 这 交换器似乎直接或间接被钙调磷酸酶失活 而 PMCI 基因表达似乎是由钙调神经磷酸酶显着诱导的。 该提案旨在定义钙调神经磷酸酶如何实现这些特定的 影响。 我们将克隆并表征下游的监管因素 钙调神经磷酸酶，将它们组织成功能序列，确定如何 这些因素影响目标及其对钙调神经磷酸酶的反应。 这 从这些研究中获得的结果与 来自哺乳动物细胞 Ca2+ 信号传导研究的信息。 我们还提供了新的证据表明酵母中的 Ca2+ 信号传导 生理上很重要。 我们观察到两个新的环境条件 它通过引发假定的 Ca2+ 信号来激活钙调神经磷酸酶。 钙2+ 这些发现预测的渠道和监管因素目前正在 未知。 因此，我们建议类似于电容性 Ca2+ 输入 描述了哺乳动物细胞。 我们将进行基因筛查 揭示了观察到的 Ca2+ 流入增加所需的因素 对细胞内 Ca2+ 池耗尽的反应。 为了完成这些 我们将使用专门设计的遗传、分子、细胞目标 生物学、生理学和生化技术。