Mutations in the Saccharomyces cerevisiae Kinase Cbk1p Lead to a Fertility Defect That Can Be Suppressed by the Absence of Brr1p or Mpt5p (Puf5p), Proteins Involved in RNA Metabolism

Abstract

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.

Mitotic Exit and Separation of Mother and Daughter Cells

“…Cbk1's activity fluctuates modestly over the cell cycle, with peaks occurring during bud formation and cell separation (Weiss et al 2002), but the importance of this change is not clear. Indeed, alleles that drastically compromise Cbk1's kinase activity have modest effects on the in vivo function of the Mob2-Cbk1 complex, suggesting that control of its specific activity is a relatively unimportant regulatory input (Racki et al 2000;Colman-Lerner et al 2001;Weiss et al 2002;Jansen et al 2006;Bourens et al 2009). …”

“…Genetic analysis indicates that phosphorylation of both the HM site and activation loop site is important for Cbk1 function, although to different degrees (Jansen et al 2006;Bourens et al 2009;Panozzo et al 2010). Neither modification is required for recruitment of Cbk1 to the daughter cell cortex and bud neck.…”

“…Neither modification is required for recruitment of Cbk1 to the daughter cell cortex and bud neck. Substitution of alanine at the activation loop phosphoacceptor site severely compromises Cbk1's catalytic activity, making it nearly undetectable; yet this allele has a notably mild phenotype, exhibiting moderate defects in polarized growth, mating, and cell separation (Jansen et al 2006;Bourens et al 2009). In contrast, replacing the phosphoacceptor threonine at Cbk1's HM site position completely abolishes its in vivo function, but not the in vitro kinase activity of Mob2-Cbk1 immunoprecipitated from asynchronous cells.…”

The RAM Network in Pathogenic Fungi

“…The RAM network has been most extensively studied in S. cerevisiae and has been implicated in the regulation of a number of cellular processes, including mother-daughter cell separation (3,18,65,69), daughter cell-specific gene transcription (12,81), maintenance of cell wall integrity (18,50,77), cell cycle progression (4,7,70), mating projection formation and mating efficiency (3,5,65,81), polarized growth (18,65,69,81), and stress signaling (47). As detailed in this review, many of the functions of the RAM pathway characterized in S. cerevisiae are conserved in other model and pathogenic fungi.…”

“…Genetic and biochemical experiments indicate that ScCbk1 kinase activity and function are dependent on each of the other RAM proteins as well as phosphorylation at two sites that are highly conserved among Ndr/Lats family kinases (5,38,65,67). The details and consequences of ScCbk1 phosphorylation have been reviewed elsewhere and are briefly summarized here (24,56).…”