No abstract

In the yeast Saccharomyces cerevisiae, environmental stress conditions that damage the cell wall lead to activation of the so-called "compensatory mechanism," aimed at preserving cell integrity through a remodeling of this extracellular matrix. Here we used DNA microarrays to investigate the molecular basis of this response to two agents that induce transient cell wall damage; namely Congo Red and Zymolyase. Treatment of the cells with these two agents elicited the up-regulation of 132 and 101 genes respectively, the main functional groups among them being involved in cell wall construction and metabolism. The main response does not occur until hours after exposure to the cell wall-perturbing agent. In some cases, this response was transient, but more sustained in others, especially in the case of the genes involved in cell wall remodeling. Clustering of these data together with those from the response to constitutive cell wall damage, revealed the existence of a cluster of co-regulated genes that was strongly induced under all conditions assayed. Those genes induced by cell wall damage showed an enrichment in DNA binding motifs for Rlm1p, Crz1p, SBF (Swi4p/ Swi6p), Msn2p/Msn4p, Ste12p, and Tec1p transcription factors, suggesting a complex regulation of this response together with the possible involvement of several signaling pathways. With the exception of PHO89 and FKS2, none of the genes induced by Congo Red was up-regulated in a slt2 strain. Moreover, characterization of the transcriptional response to Congo Red in a rlm1 mutant strain revealed that only a few genes (i.e. PHO89, FKS2, YLR042C, and CHA1) were induced at least partially independently of the transcription factor Rlm1p, the rest being totally dependent on this transcription factor for their activation. Our findings consistently demonstrate that the cell integrity signaling pathway regulates the cell wall damage compensatory response, mainly through transcriptional activation mediated by Rlm1p.

Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity. To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e. fks1, kre6, mnn9, gas1, and knr4 mutants) that affects in a different manner the cell wall construction. Altogether, 300 responsive genes were kept based on high stringency criteria during data processing. Functional classification of these differentially expressed genes showed a substantial subset of induced genes involved in cell wall construction and an enrichment of metabolic, energy generation, and cell defense categories, whereas families of genes belonging to transcription, protein synthesis, and cellular growth were underrepresented. Clustering methods isolated a single group of ϳ80 up-regulated genes that could be considered as the stereotypical transcriptional response of the cell wall compensatory mechanism. The in silico analysis of the DNA upstream region of these co-regulated genes revealed pairwise combinations of DNA-binding sites for transcriptional factors implicated in stress and heat shock responses (Msn2/4p and Hsf1p) with Rlm1p and Swi4p, two PKC1-regulated transcription factors involved in the activation genes related to cell wall biogenesis and G 1 /S transition. Moreover, this computational analysis also uncovered the 6-bp 5 -AGCCTC-3 CDRE (calcineurindependent response element) motif in 40% of the coregulated genes. This motif was recently shown to be the DNA binding site for Crz1p, the major effector of calcineurin-regulated gene expression in yeast. Taken altogether, the data presented here lead to the conclusion that the cell wall compensatory mechanism, as triggered by cell wall mutations, integrates three major regulatory systems: namely the PKC1-SLT2 mitogen-activated protein kinase-signaling module, the "global stress" response mediated by Msn2/4p, and the Ca 2؉ /calcineurindependent pathway. The relative importance of these regulatory systems in the cell wall compensatory mechanism is discussed.Yeast and fungi are surrounded by a cell wall that is a complex structure essential for maintenance of the cell shape, prevention of lysis, and protection against harmful environmental conditions. The yeast cell wall architecture has been determined in detail over the past decade (1). It is a layered structure that is composed of ␤-1,3-and ␤-1,6-glucan (50 -60% of the cell wall dry mass), mannoproteins (40 -50%), and chitin (2%). ␤-1,3-Glucan and chitin form a fibrillar network to which mannoproteins are anchored, mostly through ␤-1,6-glucan. Some cell wall proteins, like the PIR family, can be directly linked to ␤-1,3-glucan (reviewed in Ref.2). The cell wall is not a rigid structure, since it endures all of the changes that the cell undergoes during division, morphogenesis, and differentiation.To ensure continuous integrity of the wall in accordance with its plasticity, complex mechanisms must be operating, which need to be strictly ...

Yeast mitogen-activated protein kinase (MAPK) signaling pathways transduce external stimuli into cellular responses very precisely. The MAPKs Slt2/Mpk1 and Hog1 regulate transcriptional responses of adaptation to cell wall and osmotic stresses, respectively. Unexpectedly, we observe that the activation of a cell wall integrity (CWI) response to the cell wall damage caused by zymolyase (␤-1,3 glucanase) requires both the HOG and SLT2 pathways. Zymolyase activates both MAPKs and Slt2 activation depends on the Sho1 branch of the HOG pathway under these conditions. Moreover, adaptation to zymolyase requires essential components of the CWI pathway, namely the redundant MAPKKs Mkk1/ Mkk2, the MAPKKK Bck1, and Pkc1, but it does not require upstream elements, including the sensors and the guanine nucleotide exchange factors of this pathway. In addition, the transcriptional activation of genes involved in adaptation to cell wall stress, like CRH1, depends on the transcriptional factor Rlm1 regulated by Slt2, but not on the transcription factors regulated by Hog1. Consistent with these findings, both MAPK pathways are essential for cell survival under these circumstances because mutant strains deficient in different components of both pathways are hypersensitive to zymolyase. Thus, a sequential activation of two MAPK pathways is required for cellular adaptation to cell wall damage. INTRODUCTIONSaccharomyces cerevisiae yeast cells are exposed to rapid and extreme changes in the environment. In response to these changes, precise responses are coordinated by the cell through different mitogen-activated protein kinase (MAPK) signaling pathways. In this sense, external cues are transduced into appropriate cellular responses, allowing cells to adapt to particular environmental conditions. In budding yeast, four MAPKs, Fus3, Kss1, Hog1, and Slt2/Mpk1, control mating, filamentation/invasion, high osmolarity, and cell integrity pathways, and they are activated in response to mating pheromones, starvation, osmolarity, and cell wall damage, respectively (Qi and Elion, 2005).Yeast cell integrity depends on a particular external envelope: the cell wall, which is necessary not only for maintaining cell morphology but also for protecting cells from extreme conditions. The components of this structure form a macromolecular complex whose mechanical strength allows cells to support turgor pressure against the plasma membrane (Levin, 2005;Lesage and Bussey, 2006). Because of the importance of the cell wall for survival, stress conditions that alter this structure lead to the activation of a cellular response that has been called the "compensatory mechanism" (Popolo et al., 2001). This response is triggered by the cell in an attempt to survive, and it is characterized by 1) an increase in ␤-glucan and chitin contents; 2) changes in the association between cell wall polymers; 3) an increase in the amount of several cell wall proteins (CWPs); and 4) the relocalization of important proteins from the cell wall construction machinery to the later...

The adaptation of Saccharomyces cerevisiae to situations in which cell wall integrity is seriously compromised mainly involves the cell wall integrity (CWI) pathway. However, in a recent work (Bermejo, C., Rodriguez, E., García, R., Rodríguez-Peña, J. M., Rodríguez de la Concepción, M. L., Rivas, C., Arias, P., Nombela, C., Posas, F., and Arroyo, J. (2008) Mol. Biol. Cell 19, 1113-1124) we have demonstrated the co-participation of the high osmotic response (HOG) pathway to ensure yeast survival to cell wall stress mediated by zymolyase, which hydrolyzes the ␤-1,3 glucan network. Here we have characterized the role of both pathways in the regulation of the overall yeast transcriptional responses to zymolyase treatment using whole genome expression profiling. A main group of yeast genes is dependent on both MAPKs, Slt2 and Hog1, for their induction. The transcriptional activation of these genes depends on the MAPKKK Bck1, the transcription factor Rlm1, and elements of the sho1 branch of the HOG pathway, but not on the sensors of the CWI pathway. A second group of genes is dependent on Slt2 but not Hog1 or Pbs2. However, the induction of these genes is dependent on upstream elements of the HOG pathway such as Sho1, Ste50, and Ste11, in accordance with a sequential activation of the HOG and CWI pathways. Zymolyase also promotes an osmotic-like transcriptional response with the activation of a group of genes dependent on elements of the Sho1 branch of HOG pathway but not on Slt2, with the induction of many of them dependent on Msn2/4. Additionally, in the absence of Hog1, zymolyase induces an alternative response related to mating and filamentation as a consequence of the cross-talk between these pathways and the HOG pathway. Finally, in the absence of Slt2, zymolyase increases the induction of genes associated with osmotic adaptation with respect to the wild type, suggesting an inhibitory effect of the CWI pathway over the HOG pathway. These studies clearly reveal the complexity of the signal transduction machinery responsible for regulating yeast adaptation responses to cell wall stress.Yeast cell integrity depends on an external envelope, the cell wall, whose mechanical strength allows cells to support turgor pressure and affords them protection against extreme environmental conditions. Because this structure is essential for survival, stress conditions that alter the cell wall lead to the activation of a cellular response that allows cells to adapt and survive (2, 3). This response is mainly characterized by activation of a transcriptional adaptation program that been extensively studied in the last few years by means of DNA microarray experiments. Transcriptional responses in mutants deleted in genes that are important for cell wall biogenesis as well as those activated in the presence of cell wall-perturbing agents have been characterized (4 -7). Such responses include the transcriptional activation of specific genes for each particular stress condition but also the induction of a cluster of genes that are co-i...