PHEROMONE RESPONSE AND G PROTEINS IN YEAST

酵母属中的α和α交配型单倍体细胞 酿酒酵母分泌称为α和α因子的肽信息素， 信息素与受体或细胞的各自结合 相反的交配类型激活细胞内反应途径， 导致细胞周期停滞在细胞周期的GI期， 形态学变化和诱导基因参与了许多方面 信息素的反应和交配。 信息素的许多成分 反应途径已确定，包括受体，三个 G蛋白亚基、几种激酶和转录因子。 此外，反应途径的脱敏已被证明， 作用于途径的多个层面。 酵母系统显示出许多 类似于哺乳动物的信号转导系统。 突变 这些系统的组成部分与某些癌症和疾病有关 states. 酵母提供了一个系统，在其中可以分析这样的途径 使用遗传学和生物化学方法的组合。 拟议研究的目的是阐明以下几个方面： 这个过程 G蛋白α亚基（Scg 1）的生化测定 蛋白质将被开发，包括鸟嘌呤核苷酸的测定 解离速率，GTdR活性，和构象转变， 活跃的国家。 生化和表型的比较 改变这些活动的突变效应将阐明 G蛋白的作用机制 G α亚基的一个关键作用是 与信息素受体相互作用。 因为这两个受体 这两种相互作用的特异性 可能会有所不同。 受体结合区的突变 构建并分析表型效应以定位受体 结合结构域，并确定参与特异性的残基 与两种受体的相互作用。 等位基因特异性抑制子 这些突变将被分离，以定位受体结构域 参与G蛋白的相互作用。 在酵母途径中， G蛋白的β/γ亚基被认为与 下游效应子，尚未鉴定。 两个遗传 方法已经鉴定了效应子的几个候选基因。 将对这些基因进行表征以确定它们在该途径中的作用 并确定它们是否识别效应器。 Sst 2产品 在反应的脱敏中起着重要作用， 作用机制尚不清楚。 功能改变的Sst 2突变体是 这些突变的抑制基因将被分离出来， 确定Sst 2行动的目标。 其他实验的目的是 确定Sst 2相对于已知的 件. 最后，新的突变体被分离出来， 在这条通路上有新的负作用成分。 这些突变体 将被表征，基因将被克隆，以研究它们的 作用机制以及它们在通路中的作用位置。 这些实验 将阐明细胞反应机制的几个方面 并由此发生对信息素的脱敏。

Haploid cells of a and alpha mating type in the yeast Saccharomyces cerevisiae secrete peptide pheromones called a and alpha-factor, respective binding of the pheromones to receptors or cells of the opposite mating type activates an intracellular response pathway, which leads to cell cycle arrest in the GI phase of the cell cycle, morphological changes, and induction of genes involved in many aspects of pheromone response and mating. Many components of the pheromone response pathway have been identified, including the receptors, three subunits of a G protein, several kinases, and a transcription factor. In addition, desensitization of the response pathway has been shown to act at multiple levels of the pathway. The yeast system shows many similarities to mammalian signal transduction systems. Mutations in components of these systems are implicated in some cancer and disease states. Yeast provides a system in which such a pathway can be analyzed using a combination of genetic and biochemical approaches. The aims of the proposed research are to elucidate several aspects of this process. Biochemical assays of the alpha subunit (Scg1) of the G protein will be developed, including assays of the guanine nucleotide dissociation rate, the GTPase activity, and the conformational shift to the active state. A comparison of the biochemical and the phenotypic effects of mutations that alter these activities will elucidate the mechanism of G protein action. A critical role of the Galpha subunit is to interact with the pheromone receptors. Because the two receptors are not homologous to one another, the specificity for the two interactions may differ. Mutations in the receptor-binding domain are being constructed and analyzed for phenotypic effects to localize the receptor binding domain and to identify the residues involved in the specificity for the interactions with the two receptors. Allele-specific suppressors of these mutations will be isolated to localize the receptor domains involved in the G protein interaction. In the yeast pathway, the Beta/gamma subunit of the G protein is proposed to interact with the downstream effector, which has not been identified. Two genetic approaches have identified several candidate genes for the effector. These genes will be characterized to determine their role in the pathway and to determine whether they identify the effector. The Sst2 product plays an important role in desensitization of the response but its mechanism of action is not know. Sst2 mutants with altered function are being isolated, and suppressors of these mutations will be isolated to identify the target of Sst2 action. Other experiments are aimed at determining where in the pathway Sst2 acts with respect to known components. Finally, new mutants have been isolated that may identify new components of the pathway that play negative roles. These mutants will be characterized and the genes will be cloned to investigate their mechanism of action and where they act in the pathway. These experiments will elucidate several aspects of the mechanism by which cells respond to pheromone and by which desensitization to pheromone occurs.