The RIM101 pathway contributes to yeast cell wall assembly and its function becomes essential in the absence of mitogen-activated protein kinase Slt2p

Abstract

The Saccharomyces cerevisiae ynl294cDelta (rim21Delta) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Delta also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Delta and rim101Delta mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Delta, as well as rim101Delta, was synthetic lethal with slt2Delta, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Delta rlm1Delta and rim101Delta rlm1Delta double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.

FIG. 1.

Phenotypic characterization of the rim21Δ mutant. (A) Resistance to calcofluor on SC medium. Plates were incubated for 48 h at 28°C. (B) Chitin distribution after calcofluor (0.075 mg/ml) treatment for 3 h. Note the lower intensity of the staining in the mutant. (C) Comparative growth of wild-type and rim21Δ strains on the indicated media. 1/10 serial dilutions of overnight cultures were spotted and incubated for 48 h at 28°C. WT, wild type.

FIG. 2.

Activation of the PKC signaling pathway under different conditions. (A) Time course of Slt2p phosphorylation after calcofluor treatment. Samples were collected at the indicated times after the addition of calcofluor. The levels of total Slt2p are shown as a loading control. (B) Slt2p phosphorylation in wild-type and fks1Δ strains growing logarithmically or after calcofluor treatment. (C) Comparative levels of Slt2p phosphorylation in wild-type and rim21Δ strains after caffeine, calcofluor, or thermal stress. WT, wild type.

FIG. 3.

Phenotypic analysis of selected double mutants. Early logarithmic growing cells were spotted onto the indicated media at 1/10 serial dilutions. Growth was scored after 48 h at 28°C. Note the extreme hypersensitivity to SDS of the double fks1Δ rim21Δ mutant. WT, wild type.

FIG. 4.

Effect of the expression of the constitutive form of Rim101p. Wild-type and rim21Δ strains were transformed with pWL86 (Rim101-531p) and characterized phenotypically. (A) Resistance to calcofluor on SC medium. (B) Slt2p phosphorylation before (−) and after (+) calcofluor treatment in the indicated strains. Total Slt2p levels are shown in the lower panel. WT, wild type.

FIG. 5.

Phenotypic characterization of the smp1Δ mutants. (A) Calcofluor staining of the indicated mutants and chitin levels after calcofluor treatment measured as described in Materials and Methods. In parentheses, the relative chitin levels are indicated compared to the wild type. All pictures show identical exposure and processing times. (B) Sensitivity of the indicated strains to different drugs. WT, wild type.

FIG. 6.

Growth of the indicated strains in different media. (A) Complete tetrads grown on YEPD-1 M sorbitol were transferred in parallel to YEPD and YEPD-1 M sorbitol and incubated for 3 days at 28°C. Note the minor growth of the double mutants on plain YEPD. (B) Growth curve of the indicated strains. Logarithmically growing cells in YEPD-1 M sorbitol were diluted in the same medium to an OD₆₀₀ of 0.025, and growth was monitored for 12 h. Note the slower growth of the double rim21Δ slt2Δ mutant. WT, wild type.

FIG. 7.

Sensitivity of different strains to zymolyase. (A) Growth of indicated strains in increasing concentrations of zymolyase in YEPD media. The data are expressed as percentages of growth compared to identical cultures grown without zymolyase. See Materials and Methods for the detailed protocol. A logarithmic scale is used. (B) Same experiment as in panel A, but the cells were grown on YEPD-1 M sorbitol. Note the extreme growth reduction of the double mutant rim101Δ slt2Δ above 1,000 mU and the minor effect of zymolyase in this medium on the rest of strains. (C) Higher magnification of panel B, comparing the growth of the rim101Δ mutant and the wild type. Note the improved growth of the rim101Δ mutant in the presence of zymolyase. WT, wild type.

FIG. 8.

TEM images of the indicated strains grown in YEPD-1 M sorbitol. Cells are presented at two different magnification levels to show details. Note the less-defined outer layer, corresponding to the mannoprotein layer (B). WT, wild type. Bars, 0.4 μm.