YEAST PP2B PHOSPHATASES--ROLE IN PHEROMONE ADAPTATION

Ca 2 +/钙调蛋白依赖性磷蛋白磷酸酶（也称为PP 2B 或钙调神经磷酸酶）已经在体外被广泛表征，然而， 对它们在体内的调节活性知之甚少。 的 与钙调神经磷酸酶相关免疫抑制剂FK 506和环孢菌素A 在体外（与亲免蛋白结合）表明， 钙调神经磷酸酶可能在T细胞中发挥重要作用，这些药物 可能通过调节钙调神经磷酸酶抑制T细胞功能 活动 这项研究的目的是了解 酵母中钙调神经磷酸酶的功能，（一种简单的生物体，提供许多 实验优势），这可能会提供深入了解这一作用 更复杂的真核生物中的酶。 我们从酵母菌中纯化了一种具有钙调神经磷酸酶活性的 酿酒酵母，并分离出三个基因编码的组件，这一活动 它们是哺乳动物钙调磷酸酶亚基的同系物。 的第一个目标 本研究旨在进一步表征这些酵母钙调磷酸酶亚基， 通过研究它们的相互作用，定位和翻译后 修改，并确定它们是否是 酵母钙调磷酸酶 遗传分析表明，酵母钙调神经磷酸酶调节交配 反应 单倍体酵母细胞最初对交配信息素有反应 通过信号转导途径， 变化和G1期生长停滞，但最终细胞变得对 这种刺激，并恢复营养生长。 钙调磷酸酶突变体是 无法适应信息素信号，一旦被捕， 重新进入细胞周期。 对连续刺激的适应是许多生物的基本特性， 生物反应，包括视觉和嗅觉，因为它使 细胞对后续刺激的反应。 受体介导的适应 机制已在高等真核生物中描述，但其他模式 脱敏的可能性是存在的 抑制酵母对 已知信息素通过多种独立的机制产生，尽管 这些机制的生物化学基础大多未被表征。 到 研究钙调神经磷酸酶在适应中的作用，我们将：1）表征 信息素反应的钙调磷酸酶突变体更详细地定义， 在这种反应中，钙调神经磷酸酶起促进适应的作用，2）研究 钙调神经磷酸酶作用机制的遗传学鉴定 钙调神经磷酸酶促进适应所必需的，并使用 生物化学，以直接鉴定钙调磷酸酶的底物， 过程

Ca2+/calmodulin-dependent phosphoprotein phosphatases (also called PP2B or calcineurin) have been characterized extensively in vitro, however little is known about their regulatory activities in vivo. The immunosuppressive drugs FK506 and CyclosporinA associate with calcineurin in vitro (in conjunction with immunophilin proteins) suggesting that calcineurin may play an important role in T-cells, and that these drugs may inhibit T-cell function through their modulation of calcineurin activity. The aim of the proposed research is to understand the functions of calcineurin in yeast, (a simple organism that offers many experimental advantages), which may provide insight into the role of this enzyme in more complex eukaryotes. We purified a calcineurin-like activity from the yeast Saccharomyces cerevisiae, and isolated three genes encoding components of this activity that are homologs of mammalian calcineurin subunits. The first goal of this study is to characterize these yeast calcineurin subunits further, by examining their interactions, localizations and post-translational modifications, and determining whether they are the sole components of yeast calcineurin. Genetic analysis indicates that yeast calcineurin regulates the mating response. Haploid yeast cells initially respond to mating pheromone through a signal-transduction pathway, by undergoing morphological changes and G1 growth arrest, but eventually cells become refractory to this stimulus, and resume vegetative growth. Calcineurin mutants are unable to adapt to the pheromone signal, and once arrested, fail to reenter the cell cycle. Adaptation to a continuous stimulus is a fundamental property of many biological responses, including vision and olfaction, as it enables the cell to respond to subsequent stimuli. Receptor-mediated adaptation mechanisms have been described in higher eukaryotes, but additional modes of desensitization probably exist. Inhibition of the yeast response to pheromone is known to occur by multiple, independent mechanisms, although the biochemical bases of these mechanisms are mostly uncharacterized. To study the role of calcineurin in adaptation we will: 1) characterize the pheromone response of calcineurin mutants in more detail to define where in this response calcineurin acts to promote adaptation, and 2) study the mechanism of calcineurin action using genetics to identify components that are necessary for calcineurin-promoted adaptation, and using biochemistry to directly identify substrates of calcineurin in this process.