An Analysis of Spore Germination in Saccharomyces cerevisiae.

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

When conditions are not optimal for continued growth, eukaryotic cells stop dividing and enter into a specialized resting state known variously as G zero, the stationary phase, or the spore state. Cells in a quiescent state are often resistant to environmental stress and may remain viable, although not dividing, for very long periods of time. The transition between the period of quiescence and the mitotic cycle has been shown to be a key point of proliferative control. This project will examine a model transition, that of spore germination in the budding yeast, Saccharomyces cerevisiae. This budding yeast provides a useful background in which to carry out these studies because of the relative ease with which this organism can be manipulated in the laboratory. In addition, previous work has shown that the mechanisms regulating cell growth and division are remarkably conserved in all eukaryotes. Therefore, insights gained from these studies with yeast should be generally applicable. The experiments will further our understanding of both the regulatory mechanisms responsible for initiating germination and the molecules required for carrying out this multi-step process. A combination of genetic, biochemical and cell biological approaches will be used to achieve these goals. The specific aims of this project are as follows: 1. To characterize components required for the early steps of spore germination. Experiments will clarify the role of key signal transduction pathways in the early steps of spore germination. The Ras proteins are important regulators of cell division and are known to be required for germination. This aim will test whether an artificial activation of this signaling pathway is sufficient to trigger entry into the germination program. In addition, the experiments will test whether a second pathway, involving the Tor protein kinases, is necessary for spore germination. The Tor proteins are also essential for cell growth and good candidates for regulators of the germination process. Finally, a hypothesis suggesting that early steps in germination are controlled at the level of protein synthesis will be tested experimentally. 2. To characterize yeast mutants defective in spore germination. A full understanding of germination requires knowledge of the genes involved in this pathway. This aim will use two novel strategies to isolate and characterize yeast mutants defective in spore germination. 3. To characterize a novel enzyme required for spore uncoating. Yeast spores possess a specialized coat that must be partially removed prior to the formation of the first daughter bud following germination. This part of the project will involve the purification and characterization of an enzyme that is essential for this uncoating process, which is initiated very early in the germination program. Understanding the function of this enzyme and how it is regulated will provide important insights into the control of spore germination.