Characterization of the Yeast Cell Polarity Pathway

这个项目的长期目标是了解在真核细胞周期中控制细胞形态发生的机制。本研究的目的是揭示控制酵母细胞极性过程的分子机制。在酵母细胞周期中，响应不同信号的极化生长可以产生几种不同的形态结构，如芽、交配突起和假菌丝。我们之前已经鉴定了酵母细胞极性装置的两种蛋白质成分：ras相关的GTPase Cdc42p和鸟嘌呤核苷酸交换因子（GEF） Cdc24p。这些成分是在细胞周期中导致细胞极性产生的信号转导途径的组成部分。这些成分在其他真核生物中有保守的对应物，表明可能存在控制细胞极性的共同信号转导机制。此外，其他几个GTPase分子开关也参与了这一过程，但尚不清楚是什么信号控制了这些开关的激活和失活。该建议的具体目的是直接了解Cdc42p GTPase和Cdc24p GEF之间的分子相互作用。将要提出的问题是：1，Cdc24p和Ste20p和Cla4p蛋白激酶，Cdc42p的下游效应物，是否通过相同的Cdc42p效应域与Cdc42p相互作用？2、Cdc24p和Cdc42p的细胞定位是如何调控的？为了研究这些相互作用并进一步阐明这些蛋白在控制细胞极性方面的功能，将使用遗传和细胞生物学方法，包括酵母双杂交蛋白测定、GST融合蛋白共沉淀和突变表型的遗传表征。这些问题的答案不仅关系到对细胞生物学中信号转导机制的基本认识，而且关系到对酵母和其他真核生物细胞形态发生过程的认识。酵母细胞（Saccharomyces cerevisiae）由于其简单性，一直被用作真核细胞功能的模型。像大多数真核细胞一样，酵母细胞“知道”如何区分自身的一端（本质上是球形的）和另一端。这种“极性”调节对酵母的生命周期至关重要，但在更普遍的意义上，对大多数其他真核细胞类型的功能也至关重要。这一建议解决内部信号机制，即酵母细胞控制其自身的形态发生过程中，如交配和出芽（细胞分裂）。我们已经知道，在这一过程中涉及的一系列复杂的催化事件级联中，一系列蛋白质相互作用。这个项目将通过填充关于这个过程如何工作的重要细节来扩展我们对这个过程的知识。＊＊＊

9723071 Johnson The long-term objective of this project is to understand the mechanisms that control cellular morphogenesis during the eukaryotic cell cycle. The goal of this proposal is to decipher the molecular mechanisms that control the cell polarity process in the yeast Saccharomyces cerevisiae. Polarized growth in response to different signals during the yeast cell cycle can result in the generation of several different morphological structures, such as buds, mating projections, and pseudohyphae. We have previously characterized two protein components of the cell polarity apparatus in yeast: the Ras-related GTPase Cdc42p and it guanine-nucleotide exchange factor (GEF) Cdc24p. These components are integral parts of a signal-transduction pathway that leads to the generation of cell polarity during the cell cycle. These components have conserved counterparts in other eukaryotes, suggesting that common signal-transduction mechanism controlling cell polarity may exist. In addition, several other GTPase molecular switches have been implicated in the process, but it is unclear what signals control the activation and de-activation of these switches. The specific aims of this proposal are directed at understanding the molecular interactions between the Cdc42p GTPase and the Cdc24p GEF. The questions that will be asked are: 1, do Cdc24p and the Ste20p and Cla4p protein kinases, downstream effectors of Cdc42p, interact with Cdc42p through the same Cdc42p effector domain?; and 2, how is the cellular localization of Cdc24p and Cdc42p regulated? To study these interactions and to further elucidate the function(s) of these proteins in controlling cell polarity, genetic and cell biological approaches, including the yeast two-hybrid protein assay, GST fusion protein co-precipitation, and genetic characterization of mutant phenotypes, will be used. The answers to these questions will not only be relevant to the basic understanding of signal transduction mechanism in cell biology, but also t o the understanding of the cellular morphogenesis process in yeast and other eukaryotes. The yeast cell, Saccharomyces cerevisiae, has long been used as a model for eukaryotic cell functions because of its simplicity. Like most eukaryotic cells, the yeast cell "knows" how to distinguish one "end" of its (essentially spherical) self from another. This "polarity" regulation is critical for the yeast life cycle, but is also in a more general sense critical for the functions of most other eukaryotic cell types. This proposal addresses the internal signaling mechanisms whereby the yeast cell controls its own morphogenesis during processes such as mating and budding (cell division). It is already known that a series of proteins interact with one another in a complex cascade of catalytic events that are involved in this process. This project will extend our knowledge of this process by filling in important details about how this process works. ***