The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast. Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1. Several cell wall mutants displayed enhanced FKS2-lacZ expression, a known output of Slt2 activation. A similar response was obtained with wild-type cells grown in the presence of the cell wall perturbants Calcofluor white and Zymolyase. Upregulation of FKS2-lacZ in response to sublethal concentrations of these agents fully depended on the presence of Slt2. The same cell wall stress conditions resulted in dual threonine and tyrosine phosphorylation of Slt2. Both Slt2 phosphorylation and FKS2-lacZ induction could be largely prevented by providing osmotic support to the plasma membrane. Interestingly, Slt2 phosphorylation in response to cell wall damage required the putative plasmamembrane-located sensor Mid2 but not Hcs77/Wsc1. Finally, cell wall perturbation gave rise to cells with increased resistance to glucanase digestion and heat shock. These responses depended on the presence of Slt2. These results indicate that weakening of the cell wall activates the Slt2/Mpk1 MAP kinase pathway and results in compensatory changes in the cell wall.

SummaryLow environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to b1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to b1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with b1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to b1,3-glucan and the alkali-resistant GPI-derived linkage to b1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the b1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to b1,3-glucan was induced by low pH. The low pHinduced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1-Rho1-controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall-related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p-controlled cell wall genes and STRE-controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p-MAP kinase pathway, viz. Pkc1-R398A and Rho1-Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N) 10 GGC, was found. In Zymolyase -treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W) 4 TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold. Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.

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