REGULATION OF FILAMENTOUS GROWTH IN YEAST

本提案的目的是了解提供 特异性会聚MAP激酶途径。信息素反应 酿酒酵母中的一条途径负责传递 细胞外交配信号在单倍体接合期间进入细胞核。 它包括费洛蒙受体，一种异源三聚体G蛋白，一种高度同源的蛋白质。 保守的PAK/MEKK/MEK/MAPK激酶级联（Ste20p、Ste11p、Ste7p和 Fus3p/Kss1p）和转录因子Ste12p。要素 还需要包括Ste20p、Ste11p、Ste7p和Ste12p在内的途径 对于二倍体丝状生长（细长细胞链的发育 在氮饥饿时）和单倍体侵入生长。因此，一种激酶 级联被单倍体中的不同发育途径使用， 二倍体和输出的途径涉及相同的转录 因子，Ste 12p。这项拟议中的研究使用分子和遗传学方法， 方法来识别Ste 12p调节子和目标，并了解 Ste 12 p在丝状生长中的调节与其调节的比较 在单倍体交配中。第一个具体目标是确定和描述 丝状二倍体细胞中的ste12p调控基因。基因 Ste 12p在丝状菌生长过程中的转录调控 将用酵母lacZ融合文库克隆二倍体。他们的 表达模式，蛋白定位和突变表型将是 分析了一些克隆基因的启动子将被定位， 负责Ste 12p诱导的上游激活序列 转录。第二个目的是了解Ste12p的机制 调节丝状生长，并确定其调节剂。我们提出 研究在双链化条件下Ste12p的磷酸化。遗传 筛选被设计用于鉴定二倍体中的Ste12p激酶，因为 不需要信息素反应途径的MAP激酶Fus3p/Kss1 用于丝状生长。我们建议增加监管机构 与Stel2p一起提供二倍体特异性调节。两 将利用遗传和生物化学实验来鉴定这些 监管部门保守MAP激酶对Ste12p调控的研究 pathway解决了一个有趣的问题， 这些途径的特殊性。这项研究将加强我们的 了解MAP激酶途径在 哺乳动物细胞 遗传多态性调控的研究 易驾驭S.酿酒酵母也将进一步我们的知识，真菌 二型性，这在白色念珠菌的发病机制中很重要， 一种常见的人类真菌病原体

The aim of this proposal is to understand mechanisms that provide the specificity to converging MAP kinase pathways. The pheromone response pathway in Saccharomyces cerevisiae is responsible for transmitting extracellular mating signals into the nucleus during haploid conjugation. It includes pheromone receptors, a hetero-trimeric G protein, a highly conserved PAK/MEKK/MEK/MAPK kinase cascade (Ste20p, Ste11p, Ste7p, and Fus3p/Kss1p), and a transcription factor, Ste12p. Elements of the pathway, including Ste20p, Ste11p, Ste7p, and Ste12p, are also required for diploid filamentous growth (development of chains of elongated cells upon nitrogen starvation) and haploid invasive growth. Thus, one kinase cascade is used by different developmental pathways in haploids and diploids and the outputs of the pathways involve the same transcription factor, Ste12p. The proposed research uses molecular and genetic approaches to identify Ste12p regulators and targets, and to understand Ste 12p regulation in filamentous growth in comparison to its regulation in haploid mating. The first specific aim is to identify and characterize Ste12p regulated genes in filamentous diploid cells. Genes transcriptionally regulated by Ste12p during filamentous growth in diploids will be cloned with a yeast lacZ fusion library. Their expression pattern, protein localization and mutant phenotypes will be analyzed. Promoters of some of the cloned genes will be mapped to define the upstream activation sequences responsible for Ste 12p induced transcription. The second aim is to understand the mechanism of Ste12p regulation in filamentous growth and identify its regulators. We propose to study Ste12p phosphorylation under filamentation conditions. Genetic screens are designed to identify a Ste12p kinase in diploids because the MAP kinases Fus3p/Kss1 of the pheromone response pathway are not required for filamentous growth. We propose additional regulators functioning together with Stel2p provide the diploid specific regulation. Both genetic and biochemical experiments will be utilized to identify these regulators. Our research on Ste12p regulation by the conserved MAP kinase pathway addresses an interesting question concerning the regulation of specificity among these pathways. This study will enhance our understanding of the complicated regulation by MAP kinase pathways in mammalian cells. Investigation of dimorphic regulation in genetically tractable S. cerevisiae will also further our knowledge of fungal dimorphism, which is important in the pathogenesis of Candida albicans, a common fungal pathogen of humans.