| Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown, i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1 , a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell-cell contact where fusion occurs. In matings between Deltaprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of about 8 nm. Thus, PRM1 is the first gene demonstrated to act at the step of plasma membrane fusion during yeast mating. We used the Deltaprm1 mutant as a sensitized background in which to identify other genes important for cell fusion. We isolated mutations in KEX2 and LEM3 that impair fusion on their own and display more severe phenotypes in a Deltaprm1 background. Kex2p is a well-characterized Golgi-resident protease. It acts with Kex1p, a second protease, and a Deltakex1 mutant also displays a cell fusion defect. In Deltakex2 x WT matings, more than 80 percent of unfused mating pairs display extracellular membrane-bounded blebs at the site of cell-cell contact suggestive of a defect in membrane rearrangements during cell fusion. Lem3p is a transmembrane protein that co-localizes with Prm1p at the site of cell-cell contact. In Deltaprm1 Deltalem3 matings, fewer than 10 percent of mating pairs successfully fuse. These three genes thus provide the fist molecular handles on the process of cell-cell fusion during yeast mating. |
