YEAST PP2B PHOSPHATASES--ROLE IN PHEROMONE ADAPTATION

钙/钙调蛋白依赖的磷酸蛋白磷酸酶(又称PP2B 或钙调神经磷酸酶)已经在体外被广泛表征 对它们在体内的调控活动知之甚少。这个 与钙调神经磷酸酶相关的免疫抑制剂FK506和环孢素A 在体外(结合免疫亲和素蛋白)表明 钙调神经磷酸酶可能在T细胞中发挥重要作用，而这些药物 可能通过调节钙调神经磷酸酶抑制T细胞的功能 活动。拟议研究的目的是了解 钙调神经磷酸酶在酵母中的功能，(一种简单的有机体，提供许多 实验优势)，这可能提供对这一角色的洞察 更复杂的真核生物中的酶。 我们从酵母菌中纯化了一种钙调神经磷酸酶类似活性。 Cerevisiae，并分离出编码该活性组分的三个基因 它们是哺乳动物钙调神经磷酸酶亚单位的同源物。的第一个目标 本研究旨在进一步鉴定这些酵母钙调神经磷酸酶亚单位， 通过检查他们的互动、本地化和翻译后 修改，并确定它们是否是 酵母钙调神经磷酸酶。 遗传分析表明，酵母钙调神经磷酸酶调节交配 回应。单倍体酵母细胞最初对交配信息素的反应 通过一条信号转导途径，通过形态 变化和G1生长停滞，但最终细胞变得对 这一刺激，并恢复营养生长。钙调神经磷酸酶突变体是 无法适应信息素的信号，一旦被逮捕，就无法 重新进入细胞周期。 对持续刺激的适应是许多 生物反应，包括视觉和嗅觉，因为它使 细胞对随后的刺激做出反应。受体介导的适应 在高等真核生物中已经描述了机制，但其他模式 可能存在脱敏作用。对酵母菌反应的抑制 已知信息素是通过多种独立的机制产生的，尽管 这些机制的生化基础大多尚不明确。至 研究钙调神经磷酸酶在适应中的作用，我们将：1)表征 钙调神经磷酸酶突变体的信息素反应更详细地定义在哪里 在这一反应中，钙调神经磷酸酶起到促进适应的作用，2)研究 用遗传学方法鉴定钙调神经磷酸酶的作用机制 对于钙调神经磷酸酶促进的适应是必要的，并使用 生物化学直接鉴定钙调神经磷酸酶底物的研究 进程。

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.