The long term objective of the proposed research is to elucidate the mechanism by which the morphological changes required for the mitosis to interphase transition are regulated. Because of its excellent genetics and cytology, fission yeast is an excellent experimental system in which to study this point in the cell cycle at which the chromosomes decondense, the nuclear envelope reforms around the chromosomes, and the cells undergo cytokinesis and separate from one another. The focus of this proposal is on spi1, the evolutionarily conserved GTPase that is essential for the mitosis to interphase transition in fission yeast. spi1 is nearly identical to the human proto-oncogene p21ras in its GTP binding and hydrolysis domains, and its regulators are also evolutionarily conserved. The proposed studies will further our understanding of the function of this eukaryotic GTPase in particular, and the findings will be applicable to studies of the large family of structurally related GTPases that regulate a wide variety of cellular functions, and to the elucidation of the mechanism by which GTPases interact with their downstream targets via the activation of signal transduction pathways. Perturbations in the GTPase cycle, leading to an accumulation of either the GTP- or GDP-bound forms, have similar, easily monitored phenotypic consequences. This observation led to the generation of a collection of genes and mutants that based on a variety of criteria are likely to identify regulators and downstream targets of the spi1 GTPase. The proposed genetic, phenotypic, and biochemical analyses will elucidate their relationship to the known components of the spi1 GTPase system.
The Specific Aims of this proposal are:
SPECIFIC AIM I. Identify targets of the spi1 GTPase by characterizing the sns mutants and the med genes that interact genetically with components of the GTPase system;
SPECIFIC AIM II. Identify targets of the spi1 GTPase by isolating genes that when overproduced can rescue the temperature sensitive lethality of the pim1-d1 mutuant; and SPEICIFIC AIM III. Test the hypothesis that the nuclear envelope is a downstream target of the spi1 GTPase system.
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