A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae

Abstract

The PKC1-MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1-green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1-MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscDelta mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS-cAMP pathway. The RAS-cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscDelta strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

Figure 1

Sequence comparison of the WSC protein family. (A) Identical amino acids in at least two of the proteins are highlighted in white with a dark background. Gaps were used to maximize the alignment generated with the GCG software package. Different domains in the proteins are labeled. The conserved cysteine residues in the cysteine motif are designated by a solid circle, and the aromatic amino acids are designated by the symbol Φ. The last amino acids of this motif VY are designated by inverted solid triangles. Conserved sequences in the C terminus, KXYQ and DXXD, are boxed. (B) Schematic diagrams of the WSC family. SP, signal peptide; Cys, cysteine motif; Ser/Thr, serine and threonine-rich domain; Tm, transmembrane domain.

Figure 2

Cellular localization of WSC1. WSC1 was amplified by PCR and cloned into pMTS395 (39) to generate pMTS395-WSC1. This is a single-copy plasmid that contains a GFP (S65T) mutant gene, expressed under the control of a galactose-inducible promoter. The WSC1 gene was cloned 5′ to GFP. A functional test of this construct is shown in Fig. 6. (A) The strain KT626 was transformed with the GFP or the WSC1–GFP fusion construct. Transformants were selected on medium containing glucose and then grown for 12 h on galactose-containing medium. Cells were fixed in 4% paraformaldehyde followed by analysis using fluorescence confocal microscopy. (B) Cultures from A were diluted in glucose (glu)- or galactose (gal)-containing medium and grown for 12 h. Cells extracts were prepared and 20 μg of protein from membrane or cytosolic fractions was loaded unto a 10% gel. Immunoblot analysis was performed with a GFP monoclonal antibody (CLONTECH, product 8362–1).

Figure 3

Suppression of the lysis defect of the wscΔ strains by sorbitol. Yeast strains containing deletions of WSC1 (wsc1Δ) or WSC1 and WSC2 (wsc1Δwsc2Δ) were streaked and scored for growth after 2 days on YPD or YPD supplemented with 1 M Sorbitol at 37°C. A bck1Δ strain was used for comparison.

Figure 4

Heat shock sensitivity of a wsc1wsc2wsc3Δ mutant. Cells were grown on YPD containing 1 M sorbitol at 30°C until they reached an OD₆₀₀ of 1. Cultures were washed and resuspended in SC medium. They were shifted to 50°C for the indicated periods of time or first shifted to 37°C for 30 min followed by the shift to 50°C. After heat shock, the cells were plated on YPD containing 1 M sorbitol and scored for growth after 2–3 days at 30°C. The results are shown as percent survival relative to the cultures before treatment at 50°C. The strains are SP1, wild type, or the ALH758 wscΔ strain. Results are from three experiments.

Figure 5

Suppression of the heat shock sensitivity of the wsc1wsc2wsc3Δ strain by overexpression of IRA2 or by deletion of RAS2. (A) The wsc1wsc2wsc3Δ (ALH758) strain was transformed with IRA2 or a control plasmid. Transformants were grown for 2 days and were then shifted to 50°C for the indicated times. Cells were diluted, plated, and grown as in Fig. 4. The wild-type strain (SP1) was used for comparison. (B) The wild type (SP1), wsc1wsc2wsc3Δ (ALH758), and wsc1wsc2wsc3ras2Δ (JV758Δras) strains were grown and treated as in Fig. 4.

Figure 6

Suppression of the lysis defect of the wscΔ strains by overexpression of PKC1, RHO1, RHO3, and WSC. (A) The strain ALH715 (wsc1wsc3Δ) was transformed with various multicopy plasmids: pAD4Δ (control) (26), pIRIS7 (WSC1), pIRIS18(WSC2), pAD4Δ-WSC3, and PKC1 (3). Patches were made on selective medium with 1 M sorbitol, grown for 2 days, replica-plated onto YPD plates, and then incubated at 37°C for 3 days. The WSC3 gene (1.5 kb) was amplified by PCR and is expressed under the control of the alcohol dehydrogenase promoter (ADH). (B) The HRB718 (wsc1wsc2Δ) was transformed with plasmids expressing RHO1 or the RHO1^H68 mutant (19) under the control of a galactose-inducible promoter and the plasmids pMTS395 and pMTS395-WSC1 described in Fig. 2. Patches were made on medium containing glucose and 1 M sorbitol and after 2 days, the plates were replica-plated onto YPgal plates (yeast extract 1%/peptone 2%/galactose 2%). They were then incubated at 30°C for 3 days and scored for growth. (C) Same as in B, but the cells were transformed with plasmids expressing the mutant RHO3^A131, or RHO3^S47A131 (48).

Figure 7

Phosphorylation of MPK1 in response to a mild heat treatment in the wscΔ mutant. (A) A wild-type (ALHWT; lanes 1 and 2) or a mutant wsc1wsc2wsc3Δ (ALH758; lane 3 and 4) strain overexpressing MPK1–HA were incubated at 23°C (lanes 1 and 3) or 39°C (lanes 2 and 4) for 30 min. Phosphorylated MPK1–HA or endogenous MPK1 are marked with an arrow and were detected by immunoblotting with a phospho-specific p44/42 mitogen-activated protein kinase antibody. Lane 5 contains an untreated cell extract that is not expressing MPK1–HA. (B) Cell extracts identical to those in A were immunoblotted with the anti-HA antibody.