Characterization of the Yeast Cell Polarity Pathway

9723071约翰逊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。这项提议的目标是破译控制酿酒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导致几种不同的形态结构的产生，如芽、交配突起和假菌丝。我们以前已经鉴定了酵母细胞极性装置的两个蛋白质组分：与Ras相关的GTP酶CDc42p和它的鸟嘌呤核苷酸交换因子(Egf)CDc24p。这些成分是导致细胞周期中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具有保守的对应物，这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，但目前尚不清楚是什么信号控制了这些开关的激活和去激活。这项建议的具体目的是为了了解CDC42P GTP酶和CDC24P全球环境基金之间的分子相互作用。将会被问到的问题是：1，作为CDc42p下游效应因子的CDc24p和Ste20p和Cla4p蛋白激酶是否通过相同的CDc42p效应结构域与CDc42p相互作用？为了研究这些相互作用，并进一步阐明这些蛋白质在控制细胞极性中的功能(S)，将使用遗传和细胞生物学方法，包括酵母双杂交蛋白检测、GST融合蛋白共沉淀和突变表型的遗传特征。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理解，也关系到对酵母和其他真核生物细胞形态发生过程的理解。酵母细胞，酿酒酵母，长期以来一直被用作真核细胞功能的模型，因为它的简单。像大多数真核细胞一样，酵母细胞“知道”如何区分自己(本质上是球形的)自身的一个“末端”和另一个。这种“极性”调节对酵母的生命周期是至关重要的，但从更广泛的意义上讲，对大多数其他真核细胞类型的功能也是至关重要的。这一建议解决了酵母细胞在交配和发芽(细胞分裂)等过程中控制自身形态发生的内部信号机制。人们已经知道，一系列蛋白质在参与这一过程的一系列复杂的催化事件中相互作用。这个项目将通过填写有关这一过程如何工作的重要细节来扩展我们对这一过程的知识。***

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. ***