BIOLOGICAL FUSIONS--CONJUGATION IN YEAST

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This proposal focuses on the genetic control of morphogenesis in the fungus Saccharomyces cerevisiae. The most striking change in shape--the conversion of a round yeast cell to a long narrow filamentous form has important implications for human disease. The long thin cells continue to divide and, remaining attached, form a chain of connected cells or pseudohypha that can penetrate the surrounding medium. This dimorphic shift from yeast to filament, common to many fungi pathogenic for humans (C.albicans, D.neoformans, and H.capsulatum) can now be unraveled by applying the sophisticated genetic techniques available in Saccharomyces but lacking in its pathogens. The genes required for pseudohyphal formation PHD, will be cloned, sequenced and used to create mutations that block the conversion of the yeast to the filamentous form. The pathway for pseudohyphal growth will be reconstructed using both the naturally occurring mutations found in lab stocks (phd5,6,7 in S288C) in combination with cloned genes that cause pseudohyphal formation when over expressed. The structure of the pseudohypha in wild type and the phd mutants will be examined both by light and electron microscopy. The PHD4 gene, which caused adherence to plastic, will be analyzed by molecular and cell biological techniques and for adherence to endothelial cells, since adherence is thought to play an important role in deep tissue invasion by fungi. The second morphogenetic process to be analyzed is cell fusion during mating. High resolution time lapse microscopy yeast conjugation will be used to reconstruct the sequence of events in the fusion process. Key to the unraveling of this pathway will be the analysis of cell fusion mutants fus 1,2,4,5,6,and 7 and a gene required for nuclear fusion, BIK1. BIK1 contains distinct functional domains, the aminoterminus for microtubule association and the carboxyterminus for nuclear fusion. The dual functions of BIK1p will be dissected by using biochemical methods, cytological localization of BIK1, the binding of BIK1p to microtubules and the characterization of genes that are synthetic lethals with deltabik1 (slbl, 2 and 3). Our work focuses on the transduction of two external signals key to the activation of the fusion pathway, mating pheromone and Ca +2. Mutations in FUS3 (encoding a protein kinase required both for signal transduction and G1 arrest) that cause constitutive activation of the signal transduction pathway in the absence of pheromone will be used to determine the role of phosphorylation of FUS3p in signal transduction. Experiments are designed to determine whether these mutants are hyperphosphorylated and, is so, how FUS3p becomes phosphorylated and dephosphorylated. Genes which mediate the pheromone stimulated Ca+2 requirement, PCR, together with the genes encoding the plasma membrane (PMC1) and vacuolar calcium pumps (PMR1) will be used to reconstruct the role of calcium in the important membrane reorganizations that take place during conjugation.