| The haploid cell of the budding yeast Saccharomyces cerevisiae, is capable of undergoing conjugation with a mating partner upon pheromone exposure. Many of the proteins that mediate these processes are conserved in higher eukaryotes. Hence, the study of yeast mating provides a general picture of processes that occur in other systems that share these conserved components.;In this study, we have developed a novel sectoring screen to isolate mating defective mutants that would otherwise be missed using conventional replica plating mating assay. We also introduced a novel follow-up screen to focus on polarity deficient mutants. The sectoring assay was designed to facilitate the collection of signaling-proficient bilateral mutants. This was achieved by monitoring FUS1 induction and isogenic mating (through mating-type switching within a colony) of mutagenized cells. The mating defective mutants identified included far1, fus2, fus3, bnil, and axl1. Two previously unreported mating mutants, sfu1 and ymo25, were also identified in our search. The SFU1 gene was cloned in a separate screen in our laboratory and encoded a product implicated in polarized morphogenesis and cell fusion. YMO25 encodes a novel gene product with homologs in mouse, Drosophila melanogaster, Aspergillus nidulans, Caenorhabditis elegans and humans. YMO25 is required for bipolar budding in diploids and found to be pheromone inducible. The ymo25 strains display abnormal cell morphology in the vegetative state and show a subtle defect in forming mating projections.;Finally, a gpa1 strain with high basal signaling levels, reduced vegetative growth, pheromone insensitivity, and a mating defect was isolated in our primary sectoring screen. The characterization of this allele revealed a single base substitution in GPA1. The mutation lies in a highly conserved region in the Galpha subunit of the heterotrimeric G-protein. Based on crystallographic studies and our observations, we feel that the altered region of GPA1 is important for regulating basal signaling through guanine nucleotide (GDP/GTP) and Gbetagamma interactions. |
