Systematic Identification of Essential Genes Required for Yeast Cell Wall Integrity: Involvement of the RSC Remodelling Complex.

Conditions altering the yeast cell wall lead to the activation of an adaptive transcriptional response mainly governed by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Two high-throughput screenings were developed using the yTHC collection of yeast conditional mutant strains to systematically identify essential genes related to cell wall integrity, and those required for the transcriptional program elicited by cell wall stress. Depleted expression of 52 essential genes resulted in hypersensitivity to the dye Calcofluor white, with chromatin organization, Golgi vesicle transport, rRNA processing, and protein glycosylation processes, as the most highly representative functional groups. Via a flow cytometry-based quantitative assay using a CWI reporter plasmid, 97 strains exhibiting reduced gene-reporter expression levels upon stress were uncovered, highlighting genes associated with RNA metabolism, transcription/translation, protein degradation, and chromatin organization. This screening also led to the discovery of 41 strains displaying a basal increase in CWI-associated gene expression, including mainly putative cell wall-related genes. Interestingly, several members of the RSC chromatin remodelling complex were uncovered in both screenings. Notably, Rsc9 was necessary to regulate the gene expression of CWI-related genes both under stress and non-stress conditions, suggesting distinct requirements of the RSC complex for remodelling particular genes.

A novel connection between the Cell Wall Integrity and the PKA pathways regulates cell wall stress response in yeast.

Fungal cells trigger adaptive mechanisms to survive in situations that compromise cell wall integrity. We show here that the global transcriptional response elicited by inhibition of the synthesis of ß-1,3-glucan by caspofungin, encompasses a set of genes that are dependent on Slt2, the MAPK of the Cell Wall Integrity (CWI) pathway, and a broad group of genes regulated independently of Slt2. Genes negatively regulated by the cyclic AMP/Protein Kinase A (PKA) signaling pathway were overrepresented in the latter group. Moreover, cell wall stress mediated by inhibition of ß-1,3-glucan synthesis, but not by other cell wall interfering compounds, negatively regulated PKA signaling as indicated by the nuclear localisation of Msn2, cellular glycogen accumulation, a decrease of intracellular cAMP levels and a severe decrease in both the activation of the small GTPase Ras2 and the phosphorylation of known substrates of PKA. All these effects relied on the plasma membrane-spanning sensor of the CWI pathway Wsc1. In addition, caspofungin induced a reduction in the cytosolic pH, which was dependent on the extracellular region of Wsc1. Therefore, alterations of the ß-1,3-glucan network in the fungal cell wall, induce, through Wsc1, the activation of the CWI pathway and parallel inhibition of PKA signaling.

Activation of the yeast cell wall integrity MAPK pathway by zymolyase depends on protease and glucanase activities and requires the mucin-like protein Hkr1 but not Msb2.

Yeast adaptation to conditions in which cell wall integrity is compromised mainly relies on the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Zymolyase, a mixture of cell wall-digesting enzymes, triggers a peculiar signaling mechanism in which activation of the CWI pathway is dependent on the high-osmolarity glycerol MAPK pathway. We have identified inhibitors of the principal enzyme activities present in zymolyase and tested their effect on the activation of the MAPK of the CWI pathway, Slt2/Mpk1. Eventually, only ß-1,3-glucanase and protease activities were essential to elicit Slt2 activation and confer lytic power to zymolyase. Moreover, we show that the osmosensor Hkr1 is required for signaling, being the most upstream element identified to date.

Chromatin remodeling by the SWI/SNF complex is essential for transcription mediated by the yeast cell wall integrity MAPK pathway.

In Saccharomyces cerevisiae, the transcriptional program triggered by cell wall stress is coordinated by Slt2/Mpk1, the mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, and is mostly mediated by the transcription factor Rlm1. Here we show that the SWI/SNF chromatin-remodeling complex plays a critical role in orchestrating the transcriptional response regulated by Rlm1. swi/snf mutants show drastically reduced expression of cell wall stress-responsive genes and hypersensitivity to cell wall-interfering compounds. On stress, binding of RNA Pol II to the promoters of these genes depends on Rlm1, Slt2, and SWI/SNF. Rlm1 physically interacts with SWI/SNF to direct its association to target promoters. Finally, we observe nucleosome displacement at the CWI-responsive gene MLP1/KDX1, which relies on the SWI/SNF complex. Taken together, our results identify the SWI/SNF complex as a key element of the CWI MAPK pathway that mediates the chromatin remodeling necessary for adequate transcriptional response to cell wall stress.

Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: novel insights into diverse MAPK outcomes.

BACKGROUND: The yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on two aspects that are only partially understood, namely, the characterization of the gene functional catalog associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional outcomes. RESULTS: A systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified. CONCLUSION: We provide new insights into yeast genes related to the CWI pathway and into how the state of activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic MAPKs.

A yeast strain biosensor to detect cell wall-perturbing agents.

The cell wall is an essential, unique and highly conserved structure in fungi, thus representing an ideal set of targets for antifungal drugs. In the model yeast S. cerevisiae, the Pkc1-mediated cell integrity signalling pathway is essential for maintenance of the cell wall. Adaptation to cell wall stress involves the transcriptional activation of genes functionally relevant for cell wall remodelling. One of these activated genes, namely MLP1/YKL161c, is an ideal indicator of cell wall perturbations, Mlp1p, being almost undetectable under normal growth conditions, accumulated in large amounts when cell wall integrity was compromised. We have developed a reporter system based on the expression of the nourseothricin resistance gene under the control of the regulatory sequences of MLP1. Yeast cells transformed with this reporter construct, subjected to a cell wall stress, by chemical agents present in the culture medium, attained a high level of nourseothricin-resistance with respect to non-stressed cells, as a consequence of increased MLP1 expression. A genetically modified S. cerevisiae strain (AT-1) including the reporter system integrated into the native MLP1 chromosomal locus was also developed. This strain was tested against several compounds, grouping different mechanisms of yeast growth inhibition, responding specifically to cell wall-perturbing agents. Our results demonstrate the usefulness and feasibility of the AT-1 strain as a biosensor to perform high-throughput antifungal screenings for the identification of antifungal compounds active on the cell wall.