Cell extensions in pkc1 mutants of Saccharomyces cerevisiae

To obtain information on cell wall synthesis and its relationship to morphology, we examined the induction of cell extensions of yeast upon the addition of isoamyl alcohol in osmotically fragile mutants that had mutations in genes related to the cell integrity pathway through activation of the mitogen-activated protein kinase cascade. We found that isoamyl alcohol induces cell extensions in pkc1 deletion mutants but not in mutants with mutations in genes positioned downstream or upstream of the PKC1 gene. These results suggest that Pkc1p functions not only in the integrity pathway but also in the induction. We characterized the elongated cells; many had two or more nuclei. We found no difference in cell surface structure between round and elongated cells from the results of chitin staining and cell wall extraction. Actin cytoskeleton was organized in elongated cells, as well as round cells. Cytochalasin D (0.08 mg/mL) inhibited the formation of actin cable but did not affect the induction of cell extensions.Key words: Saccharomyces cerevisiae, pkc1, isoamyl alcohol, cell extension.

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PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00146-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1685-1698 ◽

Cited By ~ 83

Author(s):

Kimberly J. Gerik ◽

Sujit R. Bhimireddy ◽

Jan S. Ryerse ◽

Charles A. Specht ◽

Jennifer K. Lodge

Keyword(s):

Cell Wall ◽

Nitrosative Stress ◽

Pathogenic Fungus ◽

Fungal Growth ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Wild Type ◽

Pathway Activation ◽

Mitogen Activated Protein ◽

Kinase Cascade

ABSTRACT Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

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Cooperative Regulation of DOG2, Encoding 2-Deoxyglucose-6-Phosphate Phosphatase, by Snf1 Kinase and the High-Osmolarity Glycerol–Mitogen-Activated Protein Kinase Cascade in Stress Responses of Saccharomyces cerevisiae

Journal of Bacteriology ◽

10.1128/jb.182.18.5121-5126.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5121-5126 ◽

Cited By ~ 21

Author(s):

Yoshiyuki Tsujimoto ◽

Shingo Izawa ◽

Yoshiharu Inoue

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Osmotic Stress ◽

Mitogen Activated Protein Kinase ◽

High Osmolarity ◽

High Osmolarity Glycerol ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Protein Kinase Cascade

ABSTRACT We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZtransposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression ofDOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels ofDOG2 gene expression were increased in amig1Δ mutant, while the derepression of DOG2was not observed in a snf1Δ mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptantspbs2Δ, hog1Δ, and snf1Δ. However, the osmotic induction was completely abolished in both thesnf1Δ pbs2Δ mutant and the snf1Δ hog1Δ mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol–mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

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The SH3-domain protein Bem1p coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae

Trends in Genetics ◽

10.1016/0168-9525(96)83870-4 ◽

1996 ◽

Vol 12 (11) ◽

pp. 462

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Kinase ◽

Cell Cycle Control ◽

Sh3 Domain ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Domain Protein ◽

Protein Kinase Cascade

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Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6517 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6517-6525 ◽

Cited By ~ 108

Author(s):

B M Buehrer ◽

B Errede

Keyword(s):

Saccharomyces Cerevisiae ◽

Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Mating Partner ◽

Kinase Activation ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Mating Pathway ◽

Transcriptional Output

Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner. The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1. In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis. Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade. Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation. Both proteins also affect the timing and extent of Mpk1 activation. This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation. Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2. Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1. This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions.

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