The Cbk1p Pathway Is Important for Polarized Cell Growth and Cell Separation in Saccharomyces cerevisiae

Abstract

During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth). The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth. Analysis of cbk1⌬ cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation. Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation. CBK1 and another gene, HYM1, were previously identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway. Deletion of HYM1 causes phenotypes similar to those observed in cbk1⌬ cells and disrupts the bud neck localization of Cbk1p. Wholegenome transcriptional analysis of cbk1⌬ suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinaseencoding gene CTS1 and the glucanase-encoding gene SCW11. The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p. Analysis of ace2⌬ cells reveals that Ace2p is required for cell separation but not for polarized growth. Our results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis. Cbk1p is most closely related to the Neurospora crassa Cot-1; Schizosaccharomyces pombe Orb6; Caenorhabditis elegans, Drosophila, and human Ndr; and Drosophila and mammalian WARTS/LATS kinases. Many Cbk1-related kinases have been shown to regulate cellular morphology.

Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation inSaccharomyces cerevisiae

“…The defects of pag1-1 cells in cell separation, cell shape, and mating projection formation were similar to the known defects of cbk1⌬ mutants (Racki et al, 2000;Bidlingmaier et al, 2001). This similarity led us to hypothesize that PAG1 and CBK1 may have related functions.…”

“…Previous studies found that cbk1⌬ had no growth defect (Racki et al, 2000;Bidlingmaier et al, 2001). However, we showed that CBK1 is essential for cell proliferation in a standard yeast strain and that the lethality of cbk1⌬ can be suppressed by disruption of the SSD1 gene.…”

“…These results suggest that PAG1 and CBK1 function in the same pathway. cbk1⌬ was shown to have reduced expression of yeast chitinase (Cts1p) and the low level of chitinase is probably a main cause of the cell separation defect (Racki et al, 2000;Bidlingmaier et al, 2001). Hence, we examined the expression levels of Cts1p in our strains with a quantitative chitinase assay (Table 4).…”

“…Because dominant mutations in ACE2 suppress the cell separation defect but not the cell shape and budding pattern defects of cbk1⌬, CBK1 probably regulates both an Ace2p-dependent pathway and an Ace2p-independent pathway (Racki et al, 2000). Whole genome transcriptional analysis of cbk1⌬ revealed that, in addition to the chitinase gene, the expression levels of many other genes participating in cell wall physiology were altered, suggesting that other transcriptional regulators be-sides Ace2p may act downstream of Cbk1p (Bidlingmaier et al, 2001).…”

“…cbk1 mutants were first isolated in a screen for mutations that reduce transcriptional repression by Sin3p (Dorland et al, 2000). Further studies showed that cbk1⌬ cells fail to degrade the septa connecting mother and daughter cells, resulting in the formation of cell aggregates in liquid cultures (Racki et al, 2000;Bidlingmaier et al, 2001). These cells also display other defects in cell morphogenesis, such as round cell shapes, random budding patterns, and abnormal mating projections.…”