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.

SummaryIn budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to b(1-3)glucan, and in the lateral wall, attached in part to b(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to b(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to b(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1D and gas1D mutants, which are impaired in cell wall synthesis. A temperature shift from 30°C to 38°C increased the proportion of chitin attached to b(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38°C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-b(1-6)glucan complex is found, was greatly enhanced at 38°C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.