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. ***
