DISSECTING THE PROTEIN-KINASE C MAP KINASE SIGNALING PATHWAY OF SACCHAROMYCES-CEREVISIAE/

The PKC1 gene of the budding yeast Saccharomyces cerevisiae encodes a homolog of the alpha, beta, and gamma isoforms of mammalian PKC that i s essential for cell growth. Loss of PKC1 function results in a cell l ysis defect that is suppressed by osmotic stabilizing agents, suggesti ng a defect in cell wall integrity. In this study, we show that Pkc1p- depleted cells develop holes in their cell walls positioned at their b ud tips, the site to which growth is focused during polarized cell gro wth. This result suggests that pkc1 mutants are deficient in the proce ss of cell wall remodeling during growth. In further support of this m odel, cells bearing a pkc1 Delta mutation, allowed to proliferate in t he presence of osmotic stabilizing agents, possessed cell walls that w ere only 60% as thick as wild-type tell walls. This diminution in cell wall material affected both the beta-glucan layer and the mannoprotei n layer. We have exploited the cell lysis defect of pkc1 mutants to id entify genes that function within the same signalling pathway at point s downstream of PKC1. These genes comprise a protein kinase cascade th at culminates in the activation of the MAP kinase homolog Mpk1p. The p roposed order of protein kinase function, based on genetic experiments , is Pkc1p to Bck1p to Mkk1/2p to Mpk1p. Consistent with the proposed model, Pkc1p selectively phosphorylates Bck1p in vitro and Mpk1p prote in kinase activity requires a functional BCK1 gene.