PWP2, A MEMBER OF THE WD-REPEAT FAMILY OF PROTEINS, IS AN ESSENTIAL SACCHAROMYCES-CEREVISIAE GENE INVOLVED IN CELL-SEPARATION

WD-repeat proteins contain four to eight copies of a conserved motif t hat usually ends with a tryptophan-aspartate (WD) dipeptide. The Sacch aromyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by no nhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. The PWP2 gene is essenti al for growth because spores carrying the pwp2 Delta 1::HIS3 disruptio n germinate before arresting growth with one or two large buds. The gr owth defect of pwp2 Delta 1::HIS3 cells was rescued by expression of P WP2 or epitope-tagged HA-PWP2 using the galactose-inducible GAL1 promo ter. In the absence of galactose, depletion of Pwp2p resulted in multi budded cells with defects in bud site selection, cytokinesis, and hydr olysis of the septal junction between mother and daughter cells. In ce ll fractionation studies, HA-Pwp2p was localized in the particulate co mponent of cell lysates, from which it would be solubulized by high sa lt and alkaline buffer but not by nonionic detergents or urea. Indirec t immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytosk eleton, where it functions in control of cell growth and separation.