mRNA Decapping Activator Pat1 Is Required for Efficient Yeast Adaptive Transcriptional Responses via the Cell Wall Integrity MAPK Pathway.

Cellular mRNA levels, particularly under stress conditions, can be finely regulated by the coordinated action of transcription and degradation processes. Elements of the 5'-3' mRNA degradation pathway, functionally associated with the exonuclease Xrn1, can bind to nuclear chromatin and modulate gene transcription. Within this group are the so-called decapping activators, including Pat1, Dhh1, and Lsm1. In this work, we have investigated the role of Pat1 in the yeast adaptive transcriptional response to cell wall stress. Thus, we demonstrated that in the absence of Pat1, the transcriptional induction of genes regulated by the Cell Wall Integrity MAPK pathway was significantly affected, with no effect on the stability of these transcripts. Furthermore, under cell wall stress conditions, Pat1 is recruited to Cell Wall Integrity-responsive genes in parallel with the RNA Pol II complex, participating both in pre-initiation complex assembly and transcriptional elongation. Indeed, strains lacking Pat1 showed lower recruitment of the transcription factor Rlm1, less histone H3 displacement at Cell Wall Integrity gene promoters, and impaired recruitment and progression of RNA Pol II. Moreover, Pat1 and the MAPK Slt2 occupied the coding regions interdependently. Our results support the idea that Pat1 and presumably other decay factors behave as transcriptional regulators of Cell Wall Integrity-responsive genes under cell wall stress conditions.

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.

Author Correction: Signalling through the yeast MAPK Cell Wall Integrity pathway controls P-body assembly upon cell wall stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Signalling through the yeast MAPK Cell Wall Integrity pathway controls P-body assembly upon cell wall stress.

Post-transcriptional control of mRNA is a key event in the regulation of gene expression. From yeast to human cells, P-bodies are cytoplasmic RNA-protein aggregates that play an essential role in this process, particularly under stress conditions. In this work, we show that in the model yeast Saccharomyces cerevisiae cell wall stress induces the formation of these structures. This effect is dependent on multiple elements in the Cell Wall Integrity (CWI) MAPK signalling pathway, a signal transduction cascade responsible for the maintenance of cell integrity under adverse environmental conditions. Remarkably, P-body assembly requires the catalytic activity of the MAPK of the pathway, Slt2/Mpk1. In accordance with the control exerted by this signalling pathway, the timing of P-body formation is similar to that of the activation of the CWI pathway. Noticeably, mRNAs whose expression is regulated by this pathway localize in P-bodies after the cell is exposed to stress following a temporal pattern coincident with CWI pathway activation. Moreover, when these mRNAs are overexpressed in a mutant background unable to form visible P-bodies, the cells show hypersensitivity to agents that interfere with cell wall integrity, supporting that they play a role in the mRNA lifecycle under stress conditions.

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.

The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast.

Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI) pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies.

Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases.

Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of ß-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-ß-d-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from ß-1,3-glucan, ß-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.

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.

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.

Characterization of sensor-specific stress response by transcriptional profiling of wsc1 and mid2 deletion strains and chimeric sensors in Saccharomyces cerevisiae.

Cell wall stress in the model yeast Saccharomyces cerevisiae is known to trigger an adaptive transcriptional response. This response is mediated by a specific MAPK cell wall integrity (CWI) signal transduction pathway and affects the expression of many genes whose products are involved in the remodeling of the cellular envelope. Cell wall damage is detected mainly by Wsc1 and Mid2, which are the dominant sensors of CWI pathway. Here, we first determined the transcriptional response to different cell stresses (Congo red, Caspofungin, and Zymolyase) in mid2Δ and wsc1Δ mutant strains using DNA microarrays. Mid2 turned out to be the main sensor involved in the detection of damage provoked by Congo Red, whereas the transcriptional response to Caspofungin is mediated almost exclusively by Wsc1. For stress caused by the degradation of cell wall glucans by Zymolyase, mid2Δ and wsc1Δ deletions show little effect, but the transcriptional response rather depends on the transmembrane protein Sho1, a component of the high-osmolarity glycerol (HOG) pathway. Using sensor chimeras of Wsc1 and Mid2 we studied the contribution of the cytoplasmic and extracellular regions of Mid2 and Wsc1 for sensing Caspofungin-cell wall stress. Genome-wide transcriptional characterization in addition to Slt2 MAPK phosphorylation and phenotypic analyses indicates an important role of the extracellular domain of Wsc1 in mediating signal specificity of this sensor to detect cell wall damage.

The high osmotic response and cell wall integrity pathways cooperate to regulate transcriptional responses to zymolyase-induced cell wall stress in Saccharomyces cerevisiae.

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 beta-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.

The sequential activation of the yeast HOG and SLT2 pathways is required for cell survival to cell wall stress.

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 (beta-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.

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.

The 'yeast cell wall chip' - a tool to analyse the regulation of cell wall biogenesis in Saccharomyces cerevisiae.

Within the field of Saccharomyces cerevisiae functional genomics, DNA microarrays have become a very useful tool to study genome-wide gene-expression changes under diverse experimental conditions. Here, the design and production of a gene microarray, called the 'yeast cell wall chip', specifically tailored to investigate cell wall functions, is described. This array has been validated and shown to be useful to address gene involvement in the regulation of the response to cell wall damage in yeast. The advantages of this tailored gene microarray, which contains 390 genes, in terms of reproducibility, accuracy, versatility and ease of use are reported. Importantly, the microarray design permits the performance of a double hybridization process (two experiments) on the same slide. Cell wall stress leads to the transcriptional activation of a set of genes involved in cell wall remodelling. This response has been shown to be strongly controlled by the MAP kinase (MAPK) Slt2p, but other signalling pathways have also been suggested to be involved in this process. Here, using the tailored microarray, the role of the HOG1 pathway in the regulation of the transcriptional compensatory response to cell wall damage was evaluated by comparing the transcriptional profiles of a hog1 mutant and a wild-type strain in the presence of Congo red. Two genes, YFL014W (HSP12) and YLR414C, were found to be dependent on the Hog1p MAPK for their induction, indicating that an additional level of regulation of cell wall functions is mediated by this MAPK.

CRR1, a gene encoding a putative transglycosidase, is required for proper spore wall assembly in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, sporulation is a developmental process that converts a single cell into four haploid spores. The four haploid nuclei are encapsulated within multilayered spore walls that protect them against stressful conditions. The formation of the spore-specific cell wall is a highly coordinated process that requires the participation of enzymic activities for biosynthesis, degradation, and cross-linking between components. Here the sporulation-specific gene CRR1, encoding a putative transglycosidase that is required for proper spore wall assembly, is described. Both the transcription of CRR1 and the synthesis of Crr1p were induced biphasically under sporulating conditions, with a first expression peak displaying kinetics similar to those of the middle to middle-late sporulation-specific genes, and a second late peak after 24 h under these conditions. Localization studies revealed that Crr1p localized to the spore wall that surrounds each of the four ascospores within the mature asci. Mutation of this gene had no effect on the efficiency of spore formation. However, crr1 mutant spores were sensitive to hydrolytic enzymes such as glusulase and to heat-shock treatments, underscoring the importance of this gene in the proper formation and assembly of the ascospore wall. Moreover, the deletion of CRR1 had additive effects with respect to the sensitivity of cda1 cda2 mutants to these treatments. Interestingly, overexpression of CRR1 not only complemented the phenotype of the crr1 strain but also rendered spores more resistant to the stress conditions than the wild-type. Like other mutants impaired in the formation of the spore outer layer, crr1 mutants were permeable to Calcofluor White. Finally, detailed analysis by electron microscopy of the spore walls in the crr1 mutants revealed a defect in the assembly of the spore wall components, suggesting a role for Crr1p in the cross-linking between the inner (glucan/mannoprotein) and the outer (chitosan/dityrosine) spore layers.

Mechanisms for targeting of the Saccharomyces cerevisiae GPI-anchored cell wall protein Crh2p to polarised growth sites.

The cell wall is an essential structure that preserves the osmotic integrity of fungal cells and determines cellular morphology during developmental programs. The high number of different wall components demands a variety of processes to deliver precursors and synthetic proteins to the proper location at the right time for wall development and modification. Here, the specificity of the mechanisms that regulate the temporal and spatial localisation of cell wall proteins to sites of polarised growth in Saccharomyces cerevisiae is investigated. For this purpose, the localisation of Crh2p, a cell wall glycosylphosphatidylinositol (GPI)-anchored mannoprotein that we have recently described as involved in cell wall construction and localised to polarised growth sites, was followed using a Crh2p-GFP fusion protein. Crh2p distribution was studied in several genetic backgrounds affected in different steps of the cell polarity establishment machinery or/and bud morphogenesis. Crh2p is localised at the mother-bud neck in bud1 cells following the random budding pattern characteristic of this mutant. The Crh2p distribution was greatly altered in a cdc42-1 mutant, indicating complete dependence on an organised actin cytoskeleton for polarised Crh2p distribution. The usual deposition of Crh2p in a ring at the base of growing buds was lacking in cdc10-11 cells growing under restrictive temperature conditions, whereas Crh2p deposition at the septum region was absent in both cdc10-11 and cdc15-lyt1 cells. These results point to the dependence of Crh2p localisation at the bud-neck on both septins and septum integrity. Furthermore, in the absence of Bni4p, a scaffold protein involved in the targeting of the chitin synthase III complex to the bud neck, Crh2p was not longer found at the neck in large-budded cells undergoing cytokinesis. Finally, Crh2p was not properly localised in cells deleted in CHS5, which encodes a protein involved in the transport of Chs3p, and was completely mislocalised in sbe2/sbe22 mutants, suggesting that the transport systems for Chs3p and Crh2p are to a certain extent coincident. The transport of other GPI-cell wall proteins, such as Cwp1p, however, does not depend on these systems as the localisation of the latter protein was not affected in either of these mutants.

Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family.

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.