G Protein Mediated Downregulation of a MAP Kinase in Yeast

收集有关环境的信息，整合这些信息，并根据变化的条件做出反应，是所有细胞所必需的基本能力。在真核生物中，检测和跨质膜传递信息的最常见策略之一依赖于与异源三聚体G蛋白偶联的七螺旋受体。发芽酵母的单倍体细胞使用受体偶联G蛋白来感知和适应相反交配型细胞分泌的交配信息素。G蛋白的G by亚基连接到一个MAP激酶级联系统，该系统的激活阻止了营养细胞的增殖，并诱导营养细胞分化为配子。不能交配的酵母细胞最终会恢复生长，即使持续暴露在信息素下也是如此。因此，酵母的交配反应途径，就像高等真核生物的类似信号系统一样，可以适应连续的信号。我们已经确定信息素响应Gα蛋白Gpa1刺激对信息素的适应，并且很可能是通过两个独立的机制实现的。首先，Gpa1被认为刺激一种未知的蛋白质结合并灭活G-AA。第二，有证据表明Gpa1抑制信息素反应的MAPK，Fus3。后一种机制既可以使Fus3失活，也可以将其从细胞核中移除。人们认为，MAP激酶进出细胞核是信号转导调节的主要方式，但对核质转运的调控方式知之甚少。最近，有人提出了一种模型，即Gpa1通过诱导Fus3的空泡定位来刺激交配信号的长期下调，并提出了调节Fus3的磷酸酶Msg5。在这项调查中，这一模型的两个最重要的原则将得到检验。遗传和生化方法将被用来确定Gpa1在信息素处理后的恢复过程中是否刺激Fus3从细胞核输出，以及Fus3是否与Msg5在复合体中移位。该项目的第二个目标是测试Gpa1抑制Fus3激酶活性的可能性，如果是的话，确定这种作用是直接通过变构相互作用介导的，还是通过Msg5间接介导的。除了测试特定的假说外，还将使用一种经过验证的方案来分离在Gpa1介导的适应中产生缺陷的FUS3的新等位基因。Fus3的突变形式将根据它们的激酶活性、定位和与Gpa1结合的能力来鉴定。这些实验的结果有望阐明Gpa1下调Fus3的机制。由于适应性机制是调节信号通路的基本手段，而且G蛋白和MAP激酶级联在所有真核细胞中发挥着重要的信号作用，因此这项工作可能会产生重要的先例。

Collecting information about the environment, integrating it and responding in accordance with changing conditions are fundamental abilities required of all cells. In eukaryotes, one of the most common strategies for detecting and transmitting information across the plasma membrane depends on heptihelical receptors coupled to heterotrimeric G proteins. Haploid cells of the budding yeast Saccharomyces cerevisiae use a receptor coupled G protein both to sense and adapt to mating pheromone secreted by cells of opposite mating type. The G By subunit of the G protein couples to a MAP kinase cascade, the activation of which blocksproliferation and induces the differentiation of vegetative cells into gametes. Yeast cells that are unable to mate eventually resume growth, even when continuously exposed to pheromone. Thus, the mating response pathway of yeast, like analogous signaling systems of higher eukaryotes, can adapt to a continuous signal. We have established that the pheromone responsive G a protein, Gpa1, stimulates adaptation to pheromone, and that most likely it does so by two independent mechanisms. First, it is thought that Gpa1 stimulates an unknown protein to bind and inactivate G aa. Second, there is evidence that Gpa1 inhibits the pheromone responsive MAP kinase, Fus3. The latter mechanism may serve both to inactivate Fus3, and to remove itfrom the nucleus. It is believed that the movement of MAP kinases into and out of the nucleus is a primary method of signal transduction regulation, but the ways in which nucleocytoplasmic transport is controlled are poorly understood. Recently, a model was posited in which Gpa1 stimulates long-term downregulation of the mating signal by inducing vacuolar localization of Fus3 and the phosphatase that regulates it, Msg5 is proposed. In this investigation, the two most important tenets of this model will be tested. Genetic and biochemical approaches will be used to determine whether Gpa1 stimulates the export of Fus3 from the nucleus during recovery from pheromone treatment, and whether Fus3 is translocated in complex with Msg5. A second goal of the project is to test the possibility that Gpa1 inhibits Fus3 kinase activity, and if so, to determine whether this effect is mediated directly, by allosteric interaction, or indirectly, via Msg5. In addition to testing specific hypotheses, a proven protocol will be used to isolate novel alleles of FUS3 that confer defects in Gpa1-mediated adaptation. The mutant forms of Fus3 will be characterized according to their kinase activity, localization, and ability to associate with Gpa1. The results of these experiments are expected to elucidate the mechanisms by which Gpa1 downregulates Fus3. Because adaptive mechanisms are a fundamental means of regulating signaling pathways, and because G proteins and MAP kinase cascades play important signaling roles in all eukaryotic cells, important precedents are likely to result from this work.