In Saccharomyces cerevisiae, the RAM network is involved in cell separation after cytokinesis, cell integrity and cell polarity. The key function of this network is the regulation of the activity of the protein kinase Cbk1p, which is a member of the conserved NDR kinase family. Cbk1p function is controlled by its sub-cellular localization and at least two phosphorylation events: an auto phosphorylation in the kinase domain (S570) and the phosphorylation of a C-terminal hydrophobic motif by an upstream kinase (T743). After a UV mutagenesis, we have isolated 115 independent extragenic suppressors of four ram mutations: tao3, hym1, kic1 and sog2. Over 50% of the suppressors affect a single residue in Cbk1p (S745F), which is close to the phosphorylation site in the hydrophobic motif. Our results show that the CBK1-S745F allele leads to a constitutively active form of Cbk1p that is independent of the upstream RAM network. We hypothesize that the mutant Cbk1-S745Fp mimics the effect of the phosphorylation of T743.
In Saccharomyces cerevisiae the protein kinase Cbk1p is a member of the regulation of Ace2p and cellular morphogenesis (RAM) network that is involved in cell separation after cytokinesis, cell integrity, and cell polarity. In cell separation, the RAM network promotes the daughter cell-specific localization of the transcription factor Ace2p, resulting in the asymmetric transcription of genes whose products are necessary to digest the septum joining the mother and the daughter cell. RAM and SSD1 play a role in the maintenance of cell integrity. In the presence of a wild-type SSD1 gene, deletion of any RAM component causes cell lysis. We show here that some mutations of CBK1 also lead to a reduced fertility and a reduced expression of some of the mating type-specific genes. As polarized growth is an integral part of the mating process, we have isolated suppressors of the fertility defect. Among these, mutations in BRR1 or MPT5 lead to a restoration of fertility and a more-or-less pronounced restoration of polarity; they also show genetic interactions with SSD1. Our experiments reveal a multilayered system controlling aspects of cell separation, cell integrity, mating, and polarized growth.
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