- Research article
- Open Access
Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: Novel insights into diverse MAPK outcomes
BMC Genomics volume 12, Article number: 390 (2011)
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.
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.
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.
The capacity to respond properly to external stimuli or environmental conditions is essential for homeostasis in eukaryotes. Signaling pathways, in particular those mediated by mitogen-activated protein kinases (MAPKs), play a key role in these processes. A significant amount of research has been conducted in recent years to characterize these signal transduction pathways, based largely on the model yeast Saccharomyces cerevisiae (also known as baker's or budding yeast). Since MAPK pathways are evolutionarily conserved, insights gained from yeast contribute to a better understanding of orthologous pathways in higher organisms. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans, consisting of three kinases that establish a sequential activation pathway by means of phosphorylation events . The regulation of MAPK signal transduction depends on a variety of mechanisms including scaffold proteins, subcellular localization of different elements and the action of protein phosphatases .
In yeast, six MAPK cascades have been identified that mediate the response to different stimuli: (i) pheromones (pheromone response pathway); (ii) nitrogen starvation (filamentous growth pathway); (iii) hyperosmolarity (high osmolarity/glycerol pathway); (iv) STE vegetative growth pathway (SVG) ; (v) nutrient starvation (spore wall assembly pathway); and (vi) cell wall stress (CWI: cell wall integrity pathway) (see an extensive review of MAPK pathways in ). The CWI pathway is essential for maintaining cellular integrity. Therefore, mutations affecting different elements of the pathway lead to cell lysis [4, 5]. The maintenance of cellular integrity and morphology, as well as the protection of the cell from adverse environmental conditions, depends on the cell wall, an essential structure that has been characterized extensively in Saccharomyces cerevisiae (reviewed in [6, 7]). It has three major components: an inner layer of glucans (β-1,3 and β-1,6-glucan), chitin and an outer layer of mannoproteins. These components must be correctly assembled in order to build a fully functional structure [6, 8, 9].
The essentiality of the cell wall for fungal viability makes it one of the most attractive targets for therapeutic intervention against fungal pathogens . Treatment with cell wall-perturbing agents such as the chitin-binding dyes Congo red and Calcofluor white or zymolyase, which degrades the β-1,3-glucan network, elicits a cellular survival response known as "compensatory mechanism" . This adaptive response includes changes in the yeast transcriptional program that we and other groups have characterized, not only in yeast mutants affected at different stages of cell wall biosynthesis , but also in wild-type yeast cells growing under different conditions causing transient cell wall damage [13–18]. The compensatory response leads to, among other effects, an increase in the amount of β-glucan and chitin, the production of several cell wall proteins and changes in the cross-linking between cell wall polymers . Although several signaling pathways contribute to the regulation of cell wall remodeling in order to ensure cell integrity, the regulation of this compensatory response is controlled mainly by the MAPK Slt2p/Mpk1p (hereafter noted as Slt2p) through the cell wall integrity pathway (for a review, see [20, 21]). The CWI pathway is regulated through the cell cycle, being also activated in response to a variety of external stimuli and morphological events that cause cell wall stress, such as heat stress, hypo-osmotic shock, mating pheromones, oxidative stress, actin depolymerization, cell wall-related mutations, cell wall-stressing agents, alkaline stress and endoplasmic reticulum (ER) stress [20, 22–25].
Several cell membrane proteins (Mid2, Wsc1-4 and Mtl1) [26–28] act as sensors of the CWI pathway. For further intracellular transduction of the activation signal, these sensors interact with the guanine nucleotide exchange factor (GEF) Rom2, activating the small GTPase Rho1, which then activates the yeast protein kinase C (Pkc1). The main role of activated Pkc1 is to trigger a MAPK module. Upon phosphorylation, the MAPKKK Bck1 activates a pair of redundant MAPKKs (Mkk1 and Mkk2), which phosphorylate the MAPK Slt2. Slt2 is a functional homolog of human ERK5 , a MAPK that is activated in response to both growth factors and physical and chemical stresses [30, 31]. The dually phosphorylated (Thr190/Tyr192) form of Slt2 activates two transcription factors: the MADS-box transcription factor Rlm1  and SBF, a heterodimeric complex of two proteins, Swi4 and Swi6, which are mainly involved in the regulation of gene expression in G1/S transition . Although Rlm1, activated through phosphorylation by Slt2 , is responsible for the transcriptional activation of the majority of the genes induced in CWI adaptation responses, a non-catalytic mechanism of transcriptional activation mediated by SBF through Slt2 has recently been described . This alternative mechanism extends the regulatory roles of the MAPK cascade.
In accordance with the complexity of the cellular processes related to cell wall homeostasis in yeast, cross-talk between distinct MAPK pathways has recently been described. This complicates the simple linear "top-down" concept of signaling pathways. For example, Slt2 is activated in response to hyperosmotic shock through the HOG1 MAPK pathway . Similarly, our group has shown that treating yeast cells with zymolyase also activates Slt2 in a Hog1 pathway-dependent manner [17, 37].
Considering that the CWI pathway is activated under cell wall stress, our working hypothesis is that yeast strains lacking genes functionally relevant for cell wall biogenesis or pathway regulation should present a constitutive activation of this route. In this paper, we describe the development of a genomic-wide screening in order to identify genes whose absence produces a functional constitutive activation of the CWI pathway of Saccharomyces cerevisiae under vegetative (non-stressed) growth conditions. As a result, we have identified, for the first time at genomic scale, a map of genes that could be functionally related to the CWI pathway. Furthermore, this report gives new insights into the link between the magnitude of MAPK activation and transcriptional induction or cell wall remodeling events. This information can be extended to pathogenic fungi, being useful for future therapeutic purposes.
Results and discussion
Large-scale identification of gene deletions that activate the cell wall integrity pathway in yeast
The CWI pathway of Saccharomyces cerevisiae, governed by the MAPK Slt2, is triggered under conditions that compromise the integrity of the cell wall. Signaling through this pathway can be monitored by taking advantage of reporters driven by Rlm1-responsive promoters. We have recently developed a transcriptional reporter system, especially suitable for large-scale studies, that potentially allows detecting the functional activation of the CWI pathway . Essentially, this system is based on a plasmid construction (pJS05) that includes a transcriptional fusion of the promoter region of the MLP1 gene, one of the main effectors of the transcriptional up-regulation response under cell wall stress, and the coding sequence of the NAT1 gene, which encodes resistance to the antibiotic nourseothricin. In other words, the expression of NAT1 is controlled by the MLP1 promoter; therefore, yeast mutants with a constitutive activation of the CWI pathway transformed with this construction should be able to grow in the presence of higher concentrations of the antibiotic than a wild-type strain.
Following this approach, we have designed a large-scale screening to identify yeast deletions associated with Slt2-driven transcriptional activation. To achieve this goal, we used the collection of haploid mutant strains in all non-essential genes of Saccharomyces cerevisiae (~4800 strains). As represented in Figure 1, we proceeded to transform this collection into a 96-well microplate format with the plasmid containing MLP1 P -NAT1, and the transformed strains were screened for their ability to grow in the presence of 300 μg/ml nourseothricin. This inhibitory concentration of antibiotic was previously determined using selected mutants in which basal levels of Slt2 activation were similar to those found in a wild-type strain (data not shown). After 48-72 hours of growth in a selective medium, 174 mutant strains with hyper-resistance to nourseothricin were identified and selected for further characterization.
Slt2 phosphorylation levels in nourseothricin-resistant mutants identified in the screening
A reliable method for monitoring signaling through the CWI pathway is to follow the activation state of the MAPK Slt2 by using commercially available antibodies that recognize the dual phosphorylation of conserved threonine and tyrosine residues within the activation loop of Slt2, analogous to Thr202/Tyr204 of mammalian p44/p42 MAP Kinase (ERK) . In order to associate the phosphorylation status of Slt2 to MLP1 up-regulation, the 174 deletant strains formerly selected were examined following this approach. After densitometric quantification of the phospho-Slt2 bands obtained by Western blotting analysis of the total protein extracts from each mutant strain, 64 mutants recorded higher Phospho-Slt2 levels (at least twofold) than the wild-type strain. The relative amount of phospho-Slt2 in these mutants was distributed over a wide range of values (from twice to thirtyfold) (Table 1). Representative examples of Slt2 activation in different mutants are shown in Figure 2. The complete data set (Western blots) for all the selected mutants is presented in Additional file 1. These data indicate different levels of CWI pathway activation for each mutant, suggesting that yeast cells modulate pathway activation as required by specific stimuli. Remarkably, more than 20% of the mutations identified in a large-scale analysis revealing synthetic lethal interaction with slt2 Δ  have also been isolated in our screening (Table 1).
As shown in Table 1, the functional categorization of mutants identified for constitutive Slt2 activation revealed the most representative functional groups according to the BIOBASE Knowledge Library Proteome and Saccharomyces Genome Database classifications, being those involved in cell wall organization and morphogenesis (28%), genes of unknown function (17%), signal transduction (17%), transport (11%) and transcription (9%). The remaining functional categories comprise mutants linked to metabolism (principally of RNA and proteins) and other cellular functions with lower representation in the screening (see Table 1). This distribution is consistent with putative inputs of the pathway, namely, cell wall alterations or regulatory proteins.
An analysis was conducted of predicted and known interactions between the whole set of genes identified in the screening using the STRING web resource (http://string-db.org) . This tool is very useful for the retrieval of an overall perspective of interacting genes/proteins. As shown in Figure 3, a large number of genes (38 out of 64) showed functional interactions between them. Interestingly, the interaction network was clustered again in the three main functional nodes (cell wall and morphogenesis, signal transduction and transcription) described above. From the large group of 18 mutants related to cell wall and morphogenesis, different subgroups can be highlighted. The first subgroup comprises seven genes (SLA1, ABP1, BUD6, VRP1, ARC18, TPM1, DMA2) related to actin cytoskeleton organization. Actin cytoskeleton disruption has been shown to activate the CWI pathway, probably due to the induction of cell wall stress, but the precise molecular mechanism by which Slt2 is stimulated has not been fully established . Moreover, related to this group we found the Doa4 deubiquitinating enzyme (Figure 3), which has been associated with cell morphology and actin cytoskeleton defects. In fact, DOA4 had a synthetic genetic interaction with SLA1. A second subgroup encompasses six mutants, including structural cell wall-related proteins. Three of them are glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface: Gas1, which is a β-1,3-glucanosyltransferase; Ecm33, which is linked to cell wall maintenance; and Yps7, an aspartyl protease. The corresponding deletant strains have severe cell wall defects that may explain their high basal Slt2 activation. In fact, the compensatory response elicited in a gas1 Δ strain has previously been characterized, and it involves a significant induction in MLP1 expression . In agreement with this, gas1 Δ and ecm33 Δ mutants have previously been shown to have a constitutively high level of Slt2 phosphorylation [44, 45]. Moreover, within this second subgroup we found Las21, an ER membrane protein involved in the synthesis of the GPI core structure. The absence of this protein leads to global cell wall defects. Functionally linked to Las21, the protein Arv1 was identified (Figure 3). This protein is involved in sphingolipid metabolism and has recently been related to the process of GPI synthesis and anchoring . Finally, two proteins related to chitin metabolism, Chs6, which is involved in chitin synthase Chs3 activity, and Chs1 (chitin synthase I), which is required for repairing the chitin septum during cytokinesis  were uncovered in our screening. The identification of these mutants is significant since, due to functional redundancy and the existence of gene families, the deletion of certain individual genes encoding cell wall-related proteins does not usually lead to observable phenotypes. A third subset includes pmt1 Δ, hoc1 Δ and alg6 Δ strains. All of them encode mannosyltransferase activities, and their selection in the screening is consistent with the finding that protein glycosylation of cell surface proteins is important for cell wall assembly [7, 47]. Interestingly, transcriptional responses to O- and N-glycosylation defects in yeast include the fingerprint of the cell wall damage transcriptional profile [48, 49]. In S. cerevisiae, mannosyltransferases are highly redundant, being included in different protein families [7, 50]. The identification of the abovementioned proteins is indicative of their specific importance in cell wall homeostasis.
Within the group of mutants involved in cellular signaling, we have identified several well-known negative regulators of Slt2 activity, such as the Rho1 GTPase activators Sac7 and Bem2, and protein phosphatases Sit4 and Msg5, which negatively regulate the pathway acting on Pkc1p and Slt2p, respectively [51, 52]. We also identified Rom2 in spite of being an activator (Rho1 GEF) of the CWI pathway and the protein kinase Ypk1, a regulator linking sphingolipid signaling and CWI pathway . The effect on Slt2 activation in a rom2 Δ strain has previously been described . These authors hypothesized that rom2 Δ mutants have a defective cell wall due to a decreased activity of the CWI pathway, and this alteration may triggers Slt2 activation through Rom2-independent mechanisms. The singling out of these mutants further validates our screening for discovering novel potential regulators of the CWI pathway. This is the case of Nbp2, Ksp1, Fpk1, Mog1 and Skn7. Nbp2 acts as a negative regulator of the HOG pathway by recruiting Ptc1 phosphatase to Hog1 , and has been involved in cortical ER inheritance via Slt2 , the protein kinase Ksp1 has recently been linked to filamentous growth in haploid yeast cells , and Fpk1 (flippase kinase 1) regulates phospholipid membrane translocation . Skn7 is a multifunctional transcription factor, as reflected by its ability to partner a variety of other transcriptional regulators under different conditions. It has previously been shown that Skn7 may be activated by Rho1p in response to cell wall stress , whereas Mog1p is a protein involved in nucleocytoplasmic transport. At the same time, Mog1 is required for optimal recruitment of Skn7 to specific gene promoters . The fact that Slt2 is hyperactivated in these mutants suggests novel connections between the CWI pathway and the cellular processes controlled by these elements.
Regarding the set of mutants related to transcription, many of the proteins identified are involved in RNA polymerase II dependent transcription controlling responses to a variety of conditions such as, heat stress (RTR1), oxidative stress (YPR115w) and anaerobic conditions (ROX1). Additionally, as visualized in the network map (Figure 3), there is a connection between the transcription factors Skn7 and Rox1, with both participating in the transcriptional response to oxidative stress . Also, we identified two components of a module of the mediator complex (Ssn3 and Ssn8), involved in the regulation of Skn7 activity . The appearance of these mutants in our study suggests a functional link between them and the CWI pathway. These insights enable an association to be made between this MAPK pathway and additional stressful cellular events. In this regard, it is also worth to mention that some of the mutants identified in our screening have previously shown altered sensitivity to osmotic stress (phm8 Δ, glg2 Δ and mrs3 Δ) or heat stress (ncl1 Δ, suv3 Δ and vps64 Δ).
A connection has been described between the CWI pathway and endoplasmic reticulum (ER) stress. When a cell encounters conditions that increase misfolded proteins, the Unfolded Protein Response (UPR) is activated to compensate for high levels of ER stress . The Slt2 MAPK pathway is activated during ER stress , while UPR is activated by signaling through the CWI pathway during cell wall stress . Moreover, a second pathway, the ER stress surveillance pathway (ERSU) independent of the UPR, has recently been linked to Slt2 activation . According to our results, in some of the selected mutants these mechanisms of Slt2 activation could be involved. In fact, Emp47 is required for the export of specific glycoprotein cargo from the endoplasmic reticulum, Sec72 is involved in targeting secretory proteins to ER, and Orm2 is a protein related to lipid homeostasis and protein quality control, being required for resistance to agents that induce UPR . In addition, Orm2 interacts with Slt2, but the biological significance of this interaction is still unknown . Finally, we found the cne1 mutant to be associated with ER quality control mechanisms. Cne1 is a calnexin homologue of Saccharomyces cerevisiae that may play a part in the degradation of misfolded glycoproteins.
The identification of deletant strains in genes whose function remains uncharacterized and those not previously associated to cell wall integrity, both recording an increase in Slt2 phosphorylation, was of special interest since they could be putatively associated with cell wall construction or regulation. To further investigate this possibility, a phenotypical analysis was conducted on the 11 deletant strains corresponding to genes of unknown function and 15 without clear cell wall phenotypes reported in yeast databases. Thus, the sensitivity to Congo red, caspofungin, hygromycin B, caffeine and SDS was determined. These compounds affect cell integrity through different modes of action, whereby the dye Congo red interferes with proper cell wall assembly , caspofungin consists of a β-1,3-glucan synthase inhibitor, hygromycin B hypersensitivity has been associated with glycosylation defects , SDS is a detergent that affects membrane stability and also, indirectly, cell wall construction (increased accessibility) , and caffeine is a substance that indirectly activates the CWI pathway in a TOR1-dependent fashion . Eventually, 15 out of 26 strains analyzed displayed altered sensitivity in at least one of the tests described (Table 2), suggesting that the activation of the CWI pathway in these mutants could be due to direct or indirect cell wall alterations. Interestingly, these mutants generally had more than one phenotype supporting the existence of relevant cell wall damage. In contrast, identification of mutants without apparent cell wall defects could be related to the possibility of CWI pathway activation by other stimuli. In this regard, the coordination under specific growth conditions between the CWI and other regulatory MAPK pathways has been extensively reported [20, 25]. Further supporting this, Harrison and colleagues  suggested that the activation of the CWI pathway by different stresses, rather than operating in a linear "top-down" manner, would provide lateral inputs that impact this regulatory pathway at different levels. Moreover, recent findings connect Slt2 MAPK to DNA damage responses .
Differences in chitin content between mutants with Slt2 activation
The yeast cell wall normally contains approximately 2% chitin. However, certain mutations affecting cell wall stability increase chitin levels to as much as 20% of total wall polymers . As this emergency response for cell wall repair is dependent on CWI pathway signaling, it prompted us to assess this response in the whole group of 64 mutants with basal activation of the pathway. Chitin content was measured by means of flow cytometry after staining the cells with the chitin-binding dye, Calcofluor white (CW). This is a reliable and sensitive method for chitin determination, since it has been established a linear relationship between fluorescence from yeast cells stained with CW measured by flow cytometry and the biochemical determination of chitin . Remarkably, 19 (~30%) strains contained more than twice the amount of chitin than the wild-type strain (Figure 4), denoting that a cell wall compensatory mechanism is triggered in these cells. As described above for Slt2 activation, a wide range of chitin content was observed, suggesting that increased deposition of this polymer is adapted to specific cellular requirements. In some of these mutants (nbp2 Δ, sla1 Δ, vrp1 Δ, ylr338w Δ, gas1 Δ and arc18 Δ) elevated chitin levels and genetic interactions with mutations involved in chitin synthesis have previously been described . In contrast, the remaining 45 mutants did not record an evident increase in CW binding. Interestingly, about half of those mutants with increased chitin levels have been related functionally to cell wall and/or morphogenesis, whereas within the group of mutants lacking significantly increased chitin deposition only 18% were assigned to this functional group. These data suggest a functional link between the chitin-related mechanism and activation of the CWI pathway by cell wall and morphogenesis alterations. Eight out of ten mutants with maximum Slt2 phosphorylation, most of them cell wall related (see Table 1), recorded higher chitin levels, reinforcing the idea that this polymer plays a key role in yeast for salvaging the cell under conditions that jeopardize cell integrity.
However, the identification of mutants with no significant variations in the amount of chitin indicates that CWI pathway activation could be due to alternative stimuli or internal inputs on this signaling route. In this regard, gat2 Δ and lea1 Δ mutants are two examples of special interest because they have strong Slt2 activation without affecting their chitin content (Table 1 and Figure 5a), despite they showed cell wall alterations. GAT2 encodes for a poorly characterized putative zinc finger transcription factor, while LEA1 gene product is involved in RNA splicing, although its null mutation shows synthetic sick interaction with several cell wall related genes, such as CHS1 or CHS5. In order to gain further insights into the origin of pathway activation in these mutants, we decided to construct double mutants deleting ROM2 or BCK1 in the gat2 Δ and lea1 Δ backgrounds. Rom2 is the major GEF for Rho1 that is responsible for relaying signals from cell surface to Rho1 for its activation [76, 77], while Bck1 is the first element of the CWI pathway MAPK module. These mutants allowed us to distinguish whether the phosphorylation of Slt2 was the result of cell wall stress sensing or otherwise took place directly through the MAPK module of the route independently of upstream elements. After investigating Slt2 phosphorylation in single (gat2 Δ and lea1 Δ) and double (gat2 Δrom2 Δ; gat2 Δbck1 Δ; lea1 Δrom2 Δ; lea1 Δbck1 Δ) mutants, it was evident that MAPK activation was fully dependent on Bck1, whereas the lack of Rom2 did not affect the Slt2 activation in gat2 Δ and lea1 Δ strains (Figure 5b). Nevertheless, participation of other Rho1 GEFs like Rom1 or Tus1 can not be ruled out. This is in contrast to the activation of the CWI pathway by the cell wall stress caused by Congo red, in which Rom2 is demanded for Slt2 activation . These results support the notion that particular cell wall alterations could trigger specific adaptive responses through the CWI pathway.
MAPK phosphorylation vs. transcriptional activation
An important aspect we wanted to address about the functioning of the CWI pathway was the association between the magnitude of Slt2 phosphorylation and the concomitant effect on gene expression. To achieve this goal, a selected group of mutants, representing different levels of Slt2 activation, were transformed with a reporter construction where the promoter region of the MLP1 gene was fused to the lacZ coding sequence (MLP1 P -lacZ) and transcriptional activation was studied under standard growth conditions by measuring β-Galactosidase activity. As shown in Figure 6a, a good correlation between the expression levels of MLP1 and Slt2 phosphorylation (Pearson's correlation coefficient of 0.8) was observed except for the mutant lea1 Δ, in which the reporter expression was significantly lower than expected from the MAPK phosphorylation status. Similar results were obtained when using the CWP1 promoter, another reporter of the CWI pathway (Figure 6b). In this regard, comparable behavior was recently described for the mutant msg5 Δ in which Slt2 phosphorylation is not associated with Rlm1-dependent transcription . On the basis of this observation, the existence of additional S. cerevisiae mutants with the same behavior cannot be ruled out.
In contrast to the group of mutants described above with increased levels of phospho-Slt2, another group of 110 mutants selected in the screening did not have detectable differences in Slt2 phosphorylation with respect to the wild-type strain. In order to confirm the nourseothricin resistance of these strains, we transformed them all with the reporter construct (MLP1 P -NAT1) and carried out minimal inhibitory concentration (MIC) assays using a microdilution method. Eventually, 38 mutant strains recorded higher antibiotic MIC values than that of the wild-type (Additional file 2), confirming the phenotype of nourseothricin resistance, whereas all the other mutants behaved the same as the wild-type strain. Bearing in mind that this phenotype was also confirmed for the 64 mutants with increased phosphorylation of Slt2, this group probably includes mutants with antibiotic resistance by non-CWI-related mechanisms, such as alternative effects on MLP1 expression or intrinsic antibiotic resistance.
To further investigate the molecular mechanism involved in the group of antibiotic resistant strains without detectable variation in phospho-Slt2 levels, the MLP1 P -lacZ reporter was used to monitor levels of expression of MLP1 in this set of mutants. For the majority of the mutants, MLP1 expression levels were low and in general higher than the wild-type strain (Figure 7a). However, mutant strains ssd1 Δ and pmt2 Δ had very high levels of MLP1 expression (Figure 7a) in spite of very slight, if any, Slt2 activation (Figure 7b). Pmt2 catalyzes the first step in O-mannosylation of target proteins  and SSD1 has been linked to cell wall integrity [80, 81]. To elucidate a possible participation of the CWI pathway in the activation of the gene expression in these mutants, we proceeded to generate pmt2 Δslt2 Δ, pmt2 Δrlm1 Δ, ssd1 Δslt2 Δ and ssd1 Δrlm1 Δ double mutants. By using these strains transformed with plasmids containing transcriptional fusions of MLP1, CWP1 and SED1 to lacZ, we were able to delimit the requirement of the CWI MAPK and its main transcription factor (Rlm1) for the observed transcriptional up-regulation. As shown in Figure 8a, gene activation in the absence of both CWI pathway elements, Slt2 or Rlm1, was completely annulled compared to pmt2 Δ and ssd1 Δ single mutants. These results point to the existence of situations where undetectable changes in MAPK activation (Figure 7b) give rise to remarkable consequences at gene expression levels. As further proof of the essentiality of Slt2 activity in the transcriptional response observed in the pmt2 Δ and ssd1 Δ strains, we took advantage of two mutant forms of MAPK Slt2. The first was a variant K54R, consisting of a mutation within the ATP-binding site, which blocks the catalytic activity of the protein. The second one (TA/YF) eliminates the phosphorylation of Slt2 by upstream MAPKKs Mkk1/Mkk2. Both alleles, borne on centromeric plasmids, were unable to restore MLP1 P -lacZ expression in pmt2 Δ slt2 Δ and ssd1 Δ slt2 Δ strains (Figure 8b), indicating that signaling through active Slt2 was imperative. These results sustain a mechanism of MAPK signaling in which high levels of transcriptional induction through Rlm1 are not necessarily associated with MAPK phosphorylation levels. This is relevant for CWI pathway-related studies since the phosphorylation status of Slt2 might not always reflect the real pathway outcomes. Further studies will be necessary to characterize the mechanism involved.
The fine and specific tuning of transduction pathways to ensure yeast cell survival under adverse environmental conditions is essential. Our study contributes significantly to a better understanding of how yeast cell responses to those conditions that jeopardize cell wall integrity or alter its regulation through the CWI pathway. This work has allowed us to identify, at genomic scale, a cluster of genes whose absence induces the transcriptional activation associated with the cell wall integrity compensatory mechanism. Increased levels of phosphorylated MAPK Slt2 were found in a large group of these mutants, in agreement with their direct or indirect association with cell integrity. In fact, the main cluster of genes within this group is related to cell wall biogenesis, morphogenesis and signal transduction. Of special interest are those genes detected by the screening that have not previously been involved in cell wall integrity, particularly those of unknown function, as well as genes related to transcription, RNA and protein metabolism and transport.
Although an increase in chitin content has been described as one of the effector mechanisms within the compensatory response, our results show that there is not always a correlation between activation of the CWI pathway and chitin levels. This effect is probably dependent on the stimuli involved. In fact, within the group of mutants with higher chitin levels, the functional group of genes related to cell wall and morphogenesis is overrepresented, suggesting a functional link between the activation of the chitin deposition-mediated mechanism and cell wall defects.
Another aspect of interest is the lack of uniformity of the magnitude of MAPK activation and the transcriptional outputs in the mutants analyzed, suggesting that their modulation could be relevant for cellular adaptation to specific circumstances. Although levels of MAPK activation generally correlate with transcriptional up-regulation, there are also, and unexpectedly, mutants with a transcriptional activation dependent on a functional Slt2 MAPK and the transcription factor Rlm1, despite not having significant levels of phospho-Slt2. Bearing in mind that this phenomenon could take place in other circumstances, MAPK pathway related studies should seek information on both MAPK activation and gene expression to discover whether the pathway plays a role under specific conditions.
Strains and media
Experiments were performed with the full collection of Saccharomyces cerevisiae strains (BY4741 background, MAT a; his3 Δ1; leu2 Δ0; met15 Δ0; ura3 Δ0) individually deleted in all of the ORFs identified in this organism (4840) that were replaced by the Geneticin resistance-codifying Kan MX4 module. This collection was provided by Euroscarf (Germany). Double mutants slt2 Δpmt2 Δ, slt2 Δssd1 Δ, rlm1 Δpmt2 Δ, rlm1 Δssd1 Δ, gat2 Δrom2 Δ, lea1 Δrom2 Δ, gat2 Δbck1 Δ and lea1 Δbck1 Δ were primarily obtained by directly replacing the genes SLT2 or RLM1 and ROM2 or BCK1 in the corresponding pmt2 Δ or ssd1 Δ and gat2 Δ or lea1 Δ single-deletant-strains, respectively, with the HIS3 marker using the SFH PCR-based method described by Wach and colleagues . Alternatively, slt2 Δ::HIS3, rlm1 Δ::HIS3, rom2 Δ::HIS3 or bck1 Δ::HIS3 strains constructed in the wild-type BY4742 background (MAT α) were crossed with the single deletants of interest to generate the corresponding heterozygous disruptants. After sporulation and tetrad analyses of these strains with standard yeast genetic techniques, haploid double-mutant segregants were selected. Correct ORF replacements were verified by PCR.
For routine cultures, S. cerevisiae was grown on YEPD (2% glucose, 2% peptone, 1% yeast extract) or SC-Ura/Leu medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% glucose and uracil/leucine drop- out mix).
The Escherichia coli strain used as plasmid host was DH5α. For selective growth, bacteria were grown on LB medium containing 100 mg/l ampicillin.
Standard procedures were used for yeast genetic and DNA manipulations.
DNA manipulation and plasmids
General DNA manipulation methods were performed using standard techniques . Whenever necessary, sequence verification of plasmid constructions was carried out on an automated 3730 DNA Analyzer (Applied Biosystems). The plasmid pJS05  contains the promoter region of the gene MLP1 (YKL161c) fused to the NAT1 ORF (encoding for resistance to the antibiotic nourseothricin). Plasmids MLP1 P -lacZ, CWP1 P -lacZ and SED1 P -lacZ including the promoter region of MLP1, CWP1 and SED1 genes fused to lacZ have been described previously . Plasmids bearing the Slt2/Mpk1 alleles TA/YF (p2190), K54R (p2193) and the wild-type gene (p2188) were previously described .
Screening for yeast mutants displaying increased basal MLP1 expression
Initially, the full collection of yeast mutant strains distributed in seventy-five 96-well microtiter plates (Nunclon) were transformed with the plasmid pJS05 following a protocol of microtiter plate transformation , using SC-Ura as selection medium. A new set of plates containing 100 μl of fresh SC-Ura medium were then inoculated with 5 μl of the original transformation cultures and allowed to grow at 28°C for 24 h to be used to inoculate the plates for the screening for MLP1 induction. Thus, 5 μl of these pre-inoculums were used to inoculate the definitive set of microtiter plates containing 95 μl of YEPD plus 300 μg/ml of nourseothricin (Werner BioAgents, Germany) per well. The plates were incubated in a static culture at 28°C for 48-72 h and growth was determined by measuring absorbance at 550 nm in each well with a microplate reader (Model 680, Bio-Rad). The parameters described above, including incubation times, inoculum size and antibiotic concentration, were previously optimized in order to prevent the growth of the wild-type strain (unaltered MLP1 expression).
Nourseothricin susceptibility testing
Selected yeast mutants and the wild-type strain, as control, were individually transformed with the plasmid pJS05 following standard yeast genetic methods. Antibiotic susceptibility assays were performed in 96-well sterile plates, filling each well with 95 μl of YPD containing decreasing (twofold) concentrations of the antibiotic nourseothricin, ranging from 500 μg/ml to 0.78 μg/ml (including a growth control without nourseothricin for each serial dilution). Finally, each well (except for sterility controls) was inoculated with 5 μl of a cell suspension containing approximately 104 cells from an exponentially growing culture corresponding to each transformed strain under evaluation (overnight growth in SC-Ura and refreshed in YEPD). The plates were incubated in a static culture at 28°C for 40 h, and growth was determined by measuring absorbance at 550 nm in each well with a microplate reader. Each experiment was performed with at least two independent transformants.
Western blot assays
Yeast cells were grown in YEPD overnight at 24°C to an optical density of 0.8-1 (OD600). The culture was then refreshed to 0.2 OD600 and grown at 24°C for 6 h. The procedures used for immunoblot analyses, including cell collection and lysis, collection of proteins, fractionation by SDS-polyacrylamide gel electrophoresis, and transfer to nitrocellulose membranes, have been described previously . Phosphorylated Slt2/Mpk1 was detected using anti-phospho-p44/p42 MAPK (thr202 /tyr204; Cell Signaling Technology, Beverly, MA). To monitor protein loading, actin levels were determined using mouse anti-actin mAb C4 (ICN Biomedicals, Aurora, OH). For the quantification of the bands from autoradiography films, densitometric analysis was performed using the Quantity One package (Bio-Rad Laboratories). For each sample (mutant strain), a fold-change of Phospho-Slt2 regarding the levels observed in the wild-type strain was calculated as the ratio of the intensity of the P-Slt2 band normalized by the amount of actin for each sample (mutant/wt). Experiments were carried out at least in duplicate.
β-galactosidase reporter assays
Yeast transformants were grown overnight in an SC-Ura or SC-Ura-Leu medium, as required, at 24°C and then the culture was refreshed in YEPD to 0.2 OD600 and grown at 24°C for 6 h. Yeast cell extracts were prepared by harvesting cells by centrifugation from 5 ml of culture. The cells were then resuspended in 250 μl of breaking buffer (100 mM Tris-HCl pH = 8, 1 mM Dithiothreitol, 20% glycerol), and glass beads (Glasperlen ca. 1 mm, Sartorius AG, Germany) were added to break cells in a Fast-Prep machine. Finally, extracts were clarified by centrifugation and protein concentrations were measured using the Bradford method. β-galactosidase assays were performed using the crude extracts obtained as described previously , scaling the protocol to a 96-well microtiter plate format. 5-10 μl of cell extract was mixed with 90-95 μl of Z buffer plus β-mercaptoethanol (0.03%) and 20 μl of o-nitrophenyl-β-D-galactopyranoside (ONPG) (4 mg/ml in Z buffer). The absorbance of the enzymatic reaction was measured at 415 nm on a microplate reader (Model 680, Bio-Rad) after at least 10 min of incubation at 30°C and the addition of 50 μl of 1M Na2CO3 to stop the reaction. β-galactosidase activity was expressed as nmoles of ONPG converted/minute/mg of protein. Experiments were performed at least in triplicate involving three independent yeast transformants.
Cell wall phenotypic analyses
Yeast cells were grown overnight at 24°C in YEPD to mid-log phase. The culture was diluted to an OD600 of around 0.2 and then incubated at 24°C in YEPD for 4 h. These cultures were subsequently diluted again to 0.2 (approximately 15 × 103 cells in 5 μl) and four fivefold dilution series were prepared. Finally, 5 μl of each dilution was spotted on to YEPD solid media containing 100 μg/ml Congo red (Merck), 12 mM caffeine (Sigma), 200 μg/ml SDS (Duchefa Biochemie), 50 μg/ml Hygromycin B (Roche), and 40 ng/ml caspofungin (kindly provided by Merck), using a multi-blot replicator (V&P Scientific, San Diego, CA). Growth was monitored on the plates after 2-3 days at 30°C.
Flow cytometry and Microscopic analysis
Yeast strains were grown overnight at 24°C in YEPD to mid-log phase. The culture was diluted to an OD600 of around 0.2 and then incubated at 24°C in YEPD for 4 h. After this time, 1 ml of cells was collected, washed with PBS and stained with Calcofluor white (Fluorescent Brightener 28, Sigma-Aldrich, St. Louis, MO) at a final concentration of 5 μg/ml for 10 min in darkness. For the flow cytometry analysis of chitin content using Calcofluor staining, cells were analyzed with a BD LSR flow cytometer (Becton Dickinson) by acquiring fluorescence through a 380 LP filter. Cell viability was monitored by staining cells with propidium iodide (0.05 mg/ml) acquiring fluorescence through a 670 LP filter. As experimental control, stained yeasts were also analyzed by fluorescence microscopy using a Nikon TE2000 fluorescence inverted microscope equipped with a CCD camera. Digital images were acquired with an Orca C4742-95-12ER camera (Hamamatsu Photonics, Japan) and processed with Aquacosmos Imaging System software.
cell wall integrity
mitogen activated protein kinase
Qi M, Elion EA: MAP kinase pathways. J Cell Sci. 2005, 118: 3569-3572. 10.1242/jcs.02470.
Chen RE, Thorner J: Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta. 2007, 1773: 1311-1340. 10.1016/j.bbamcr.2007.05.003.
Lee BN, Elion EA: The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components. Proc Natl Acad Sci USA. 1999, 96: 12679-12684. 10.1073/pnas.96.22.12679.
Torres L, Martin H, Garcia-Saez MI, Arroyo J, Molina M, Sanchez M, Nombela C: A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants. Mol Microbiol. 1991, 5: 2845-2854. 10.1111/j.1365-2958.1991.tb01993.x.
Levin DE, Bowers B, Chen CY, Kamada Y, Watanabe M: Dissecting the protein kinase C/MAP kinase signalling pathway of Saccharomyces cerevisiae. Cell Mol Biol Res. 1994, 40: 229-239.
Klis FM, Boorsma A, De Groot PW: Cell wall construction in Saccharomyces cerevisiae. Yeast. 2006, 23: 185-202. 10.1002/yea.1349.
Lesage G, Bussey H: Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006, 70: 317-343. 10.1128/MMBR.00038-05.
Cabib E, Blanco N, Grau C, Rodriguez-Pena JM, Arroyo J: Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall. Mol Microbiol. 2007, 63: 921-935.
Cabib E, Farkas V, Kosik O, Blanco N, Arroyo J, McPhie P: Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro. J Biol Chem. 2008, 283: 29859-29872. 10.1074/jbc.M804274200.
Latge JP: Tasting the fungal cell wall. Cell Microbiol. 2010, 12: 863-872. 10.1111/j.1462-5822.2010.01474.x.
Popolo L, Gualtieri T, Ragni E: The yeast cell-wall salvage pathway. Med Mycol. 2001, 39 (Suppl 1): 111-121.
Lagorce A, Hauser NC, Labourdette D, Rodriguez C, Martin-Yken H, Arroyo J, Hoheisel JD, Francois J: Genome-wide analysis of the response to cell wall mutations in the yeast Saccharomyces cerevisiae. J Biol Chem. 2003, 278: 20345-20357. 10.1074/jbc.M211604200.
Agarwal AK, Rogers PD, Baerson SR, Jacob MR, Barker KS, Cleary JD, Walker LA, Nagle DG, Clark AM: Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae. J Biol Chem. 2003, 278: 34998-35015. 10.1074/jbc.M306291200.
Boorsma A, De Nobel H, ter Riet B, Bargmann B, Brul S, Hellingwerf KJ, Klis FM: Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae. Yeast. 2004, 21: 413-427. 10.1002/yea.1109.
Garcia R, Bermejo C, Grau C, Perez R, Rodriguez-Peña JM, Francois J, Nombela C, Arroyo J: The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J Biol Chem. 2004, 279: 15183-15195. 10.1074/jbc.M312954200.
Rodriguez-Peña JM, Perez-Diaz RM, Alvarez S, Bermejo C, Garcia R, Santiago C, Nombela C, Arroyo J: The 'yeast cell wall chip' - a tool to analyse the regulation of cell wall biogenesis in Saccharomyces cerevisiae. Microbiology. 2005, 151: 2241-2249. 10.1099/mic.0.27989-0.
Garcia R, Rodriguez-Peña JM, Bermejo C, Nombela C, Arroyo J: The high osmotic response and cell wall integrity pathways cooperate to regulate transcriptional responses to zymolyase-induced cell wall stress in Saccharomyces cerevisiae. J Biol Chem. 2009, 284: 10901-10911. 10.1074/jbc.M808693200.
Bermejo C, Garcia R, Straede A, Rodriguez-Peña JM, Nombela C, Heinisch JJ, Arroyo J: Characterization of sensor-specific stress response by transcriptional profiling of wsc1 and mid2 deletion strains and chimeric sensors in Saccharomyces cerevisiae. OMICS. 2010, 14: 679-688. 10.1089/omi.2010.0060.
Smits GJ, Van Den EH, Klis FM: Differential regulation of cell wall biogenesis during growth and development in yeast. Microbiology. 2001, 147: 781-794.
Levin DE: Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2005, 69: 262-291. 10.1128/MMBR.69.2.262-291.2005.
Heinisch JJ: Baker's yeast as a tool for the development of antifungal kinase inhibitors--targeting protein kinase C and the cell integrity pathway. Biochim Biophys Acta. 2005, 1754: 171-182.
Bonilla M, Cunningham KW: Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast. Mol Biol Cell. 2003, 14: 4296-4305. 10.1091/mbc.E03-02-0113.
Chen Y, Feldman DE, Deng C, Brown JA, De Giacomo AF, Gaw AF, Shi G, Le QT, Brown JM, Koong AC: Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae. Mol Cancer Res. 2005, 3: 669-677. 10.1158/1541-7786.MCR-05-0181.
Serrano R, Martin H, Casamayor A, Arino J: Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway. J Biol Chem. 2006, 281: 39785-39795. 10.1074/jbc.M604497200.
Rodriguez-Peña JM, Garcia R, Nombela C, Arroyo J: The high-osmolarity glycerol (HOG) and cell wall integrity (CWI) signalling pathways interplay: a yeast dialogue between MAPK routes. Yeast. 2010, 27: 495-502. 10.1002/yea.1792.
Verna J, Lodder A, Lee K, Vagts A, Ballester R: A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1997, 94: 13804-13809. 10.1073/pnas.94.25.13804.
Ketela T, Green R, Bussey H: Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J Bacteriol. 1999, 181: 3330-3340.
Rajavel M, Philip B, Buehrer BM, Errede B, Levin DE: Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae. Mol Cell Biol. 1999, 19: 3969-3976.
Truman AW, Millson SH, Nuttall JM, King V, Mollapour M, Prodromou C, Pearl LH, Piper PW: Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase. Eukaryot Cell. 2006, 5: 1914-1924. 10.1128/EC.00263-06.
Abe J, Kusuhara M, Ulevitch RJ, Berk BC, Lee JD: Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J Biol Chem. 1996, 271: 16586-16590. 10.1074/jbc.271.28.16586.
Yan C, Luo H, Lee JD, Abe J, Berk BC: Molecular cloning of mouse ERK5/BMK1 splice variants and characterization of ERK5 functional domains. J Biol Chem. 2001, 276: 10870-10878. 10.1074/jbc.M009286200.
Watanabe Y, Takaesu G, Hagiwara M, Irie K, Matsumoto K: Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol. 1997, 17: 2615-2623.
Baetz K, Moffat J, Haynes J, Chang M, Andrews B: Transcriptional coregulation by the cell integrity mitogen-activated protein kinase Slt2 and the cell cycle regulator Swi4. Mol Cell Biol. 2001, 21: 6515-6528. 10.1128/MCB.21.19.6515-6528.2001.
Jung US, Sobering AK, Romeo MJ, Levin DE: Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase. Mol Microbiol. 2002, 46: 781-789. 10.1046/j.1365-2958.2002.03198.x.
Kim KY, Truman AW, Levin DE: Yeast Mpk1 mitogen-activated protein kinase activates transcription through Swi4/Swi6 by a noncatalytic mechanism that requires upstream signal. Mol Cell Biol. 2008, 28: 2579-2589. 10.1128/MCB.01795-07.
Garcia-Rodriguez LJ, Valle R, Duran A, Roncero C: Cell integrity signaling activation in response to hyperosmotic shock in yeast. FEBS Lett. 2005, 579: 6186-6190. 10.1016/j.febslet.2005.10.001.
Bermejo C, Rodriguez E, Garcia R, Rodriguez-Peña JM, Rodriguez de la Concepcion ML, Rivas C, Arias P, Nombela C, Posas F, Arroyo J: The sequential activation of the yeast HOG and SLT2 pathways is required for cell survival to cell wall stress. Mol Biol Cell. 2008, 19: 1113-1124.
Rodriguez-Peña JM, Diez-Muñiz S, Nombela C, Arroyo J: A yeast strain biosensor to detect cell wall-perturbing agents. J Biotechnol. 2008, 133: 311-317. 10.1016/j.jbiotec.2007.10.006.
Martin H, Rodriguez-Pachon JM, Ruiz C, Nombela C, Molina M: Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem. 2000, 275: 1511-1519. 10.1074/jbc.275.2.1511.
Tong AH, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, et al: Global mapping of the yeast genetic interaction network. Science. 2004, 303: 808-813. 10.1126/science.1091317.
Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, Doerks T, Stark M, Muller J, Bork P, et al: The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011, 39: D561-D568. 10.1093/nar/gkq973.
Harrison JC, Zyla TR, Bardes ES, Lew DJ: Stress-specific activation mechanisms for the "cell integrity" MAPK pathway. J Biol Chem. 2004, 279: 2616-2622.
Fiorani P, Reid RJ, Schepis A, Jacquiau HR, Guo H, Thimmaiah P, Benedetti P, Bjornsti MA: The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons. J Biol Chem. 2004, 279: 21271-21281. 10.1074/jbc.M312338200.
De Nobel H, Ruiz C, Martin H, Morris W, Brul S, Molina M, Klis FM: Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance. Microbiology. 2000, 146: 2121-2132.
De Groot PW, Ruiz C, Vazquez de Aldana CR, Duenas E, Cid VJ, del Rey F, Rodriguez-Peña JM, Perez P, Andel A, Caubin J, et al: A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics. 2001, 2: 124-142. 10.1002/cfg.85.
Kajiwara K, Watanabe R, Pichler H, Ihara K, Murakami S, Riezman H, Funato K: Yeast ARV1 is required for efficient delivery of an early GPI intermediate to the first mannosyltransferase during GPI assembly and controls lipid flow from the endoplasmic reticulum. Mol Biol Cell. 2008, 19: 2069-2082. 10.1091/mbc.E07-08-0740.
Lommel M, Strahl S: Protein O-mannosylation: conserved from bacteria to humans. Glycobiology. 2009, 19: 816-828. 10.1093/glycob/cwp066.
Cullen PJ, Xu-Friedman R, Delrow J, Sprague GF: Genome-wide analysis of the response to protein glycosylation deficiency in yeast. FEMS Yeast Res. 2006, 6: 1264-1273. 10.1111/j.1567-1364.2006.00120.x.
Arroyo J, Hutzler J, Bermejo C, Ragni E, Garcia-Cantalejo J, Botias P, Piberger H, Schott A, Sanz AB, Strahl S: Functional and genomic analyzes of blocked protein O-mannosylation in baker's yeast. Mol Microbiol. 2011, 79: 1529-1546. 10.1111/j.1365-2958.2011.07537.x.
Willer T, Valero MC, Tanner W, Cruces J, Strahl S: O-mannosyl glycans: from yeast to novel associations with human disease. Curr Opin Struct Biol. 2003, 13: 621-630. 10.1016/j.sbi.2003.09.003.
De la Torre-Ruiz A, Torres J, Arino J, Herrero E: Sit4 is required for proper modulation of the biological functions mediated by Pkc1 and the cell integrity pathway in Saccharomyces cerevisiae. J Biol Chem. 2002, 277: 33468-33476. 10.1074/jbc.M203515200.
Flandez M, Cosano IC, Nombela C, Martin H, Molina M: Reciprocal regulation between Slt2 MAPK and isoforms of Msg5 dual-specificity protein phosphatase modulates the yeast cell integrity pathway. J Biol Chem. 2004, 279: 11027-11034. 10.1074/jbc.M306412200.
Schmelzle T, Helliwell SB, Hall MN: Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol Cell Biol. 2002, 22: 1329-1339. 10.1128/MCB.22.5.1329-1339.2002.
Lorberg A, Jacoby JJ, Schmitz HP, Heinisch JJ: The PH domain of the yeast GEF Rom2p serves an essential function in vivo. Mol Genet Genomics. 2001, 266: 505-513. 10.1007/s004380100579.
Mapes J, Ota IM: Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway. EMBO J. 2004, 23: 302-311. 10.1038/sj.emboj.7600036.
Du Y, Walker L, Novick P, Ferro-Novick S: Ptc1p regulates cortical ER inheritance via Slt2p. EMBO J. 2006, 25: 4413-4422. 10.1038/sj.emboj.7601319.
Bharucha N, Ma J, Dobry CJ, Lawson SK, Yang Z, Kumar A: Analysis of the yeast kinome reveals a network of regulated protein localization during filamentous growth. Mol Biol Cell. 2008, 19: 2708-2717. 10.1091/mbc.E07-11-1199.
Nakano K, Yamamoto T, Kishimoto T, Noji T, Tanaka K: Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry. Mol Biol Cell. 2008, 19: 1783-1797. 10.1091/mbc.E07-07-0646.
Alberts AS, Bouquin N, Johnston LH, Treisman R: Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7. J Biol Chem. 1998, 273: 8616-8622. 10.1074/jbc.273.15.8616.
Lu JM, Deschenes RJ, Fassler JS: Role for the Ran binding protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 signal transduction. Eukaryot Cell. 2004, 3: 1544-1556. 10.1128/EC.3.6.1544-1556.2004.
Kelley R, Ideker T: Genome-wide fitness and expression profiling implicate Mga2 in adaptation to hydrogen peroxide. PLoS Genet. 2009, 5: e1000488-10.1371/journal.pgen.1000488.
Lenssen E, Azzouz N, Michel A, Landrieux E, Collart MA: The Ccr4-not complex regulates Skn7 through Srb10 kinase. Eukaryot Cell. 2007, 6: 2251-2259. 10.1128/EC.00327-06.
Ron D, Walter P: Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007, 8: 519-529. 10.1038/nrm2199.
Scrimale T, Didone L, Mesy Bentley KL, Krysan DJ: The unfolded protein response is induced by the cell wall integrity mitogen-activated protein kinase signaling cascade and is required for cell wall integrity in Saccharomyces cerevisiae. Mol Biol Cell. 2009, 20: 164-175. 10.1091/mbc.E08-08-0809.
Babour A, Bicknell AA, Tourtellotte J, Niwa M: A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance. Cell. 2010, 142: 256-269. 10.1016/j.cell.2010.06.006.
Han S, Lone MA, Schneiter R, Chang A: Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control. Proc Natl Acad Sci USA. 2010, 107: 5851-5856. 10.1073/pnas.0911617107.
Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y: A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA. 2001, 98: 4569-4574. 10.1073/pnas.061034498.
Roncero C, Duran A: Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol. 1985, 163: 1180-1185.
Dean N: Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci USA. 1995, 92: 1287-1291. 10.1073/pnas.92.5.1287.
Igual JC, Johnson AL, Johnston LH: Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. EMBO J. 1996, 15: 5001-5013.
Kuranda K, Leberre V, Sokol S, Palamarczyk G, Francois J: Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol. 2006, 61: 1147-1166. 10.1111/j.1365-2958.2006.05300.x.
Bandyopadhyay S, Mehta M, Kuo D, Sung MK, Chuang R, Jaehnig EJ, Bodenmiller B, Licon K, Copeland W, Shales M, et al: Rewiring of genetic networks in response to DNA damage. Science. 2010, 330: 1385-1389. 10.1126/science.1195618.
Klis FM, Mol P, Hellingwerf K, Brul S: Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev. 2002, 26: 239-256. 10.1111/j.1574-6976.2002.tb00613.x.
Hector RF, Braun PC, Hart JT, Kamarck ME: The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans. J Med Vet Mycol. 1990, 28: 51-57. 10.1080/02681219080000071.
Lesage G, Shapiro J, Specht CA, Sdicu AM, Menard P, Hussein S, Tong AH, Boone C, Bussey H: An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genet. 2005, 6: 8-
Bickle M, Delley PA, Schmidt A, Hall MN: Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J. 1998, 17: 2235-2245. 10.1093/emboj/17.8.2235.
Philip B, Levin DE: Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol Cell Biol. 2001, 21: 271-280. 10.1128/MCB.21.1.271-280.2001.
Marin MJ, Flandez M, Bermejo C, Arroyo J, Martin H, Molina M: Different modulation of the outputs of yeast MAPK-mediated pathways by distinct stimuli and isoforms of the dual-specificity phosphatase Msg5. Mol Genet Genomics. 2009, 281: 345-359. 10.1007/s00438-008-0415-5.
Lussier M, Gentzsch M, Sdicu AM, Bussey H, Tanner W: Protein O-glycosylation in yeast. The PMT2 gene specifies a second protein O-mannosyltransferase that functions in addition to the PMT1-encoded activity. J Biol Chem. 1995, 270: 2770-2775. 10.1074/jbc.270.6.2770.
Kaeberlein M, Guarente L: Saccharomyces cerevisiae MPT5 and SSD1 function in parallel pathways to promote cell wall integrity. Genetics. 2002, 160: 83-95.
Jansen JM, Wanless AG, Seidel CW, Weiss EL: Cbk1 regulation of the RNA-binding protein Ssd1 integrates cell fate with translational control. Curr Biol. 2009, 19: 2114-2120. 10.1016/j.cub.2009.10.071.
Wach A, Brachat A, Alberti-Segui C, Rebischung C, Philippsen P: Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisia e. Yeast. 1997, 13: 1065-1075. 10.1002/(SICI)1097-0061(19970915)13:11<1065::AID-YEA159>3.0.CO;2-K.
Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A laboratory manual. 1989, Cold Spring Harbor, N.Y.: Cold Spring Laboratory Press
Gietz RD, Woods RA: Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 2002, 350: 87-96.
Amberg DC, Burke DJ, Strathern JN: Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. 2005, John Inglis, Cold Spring Harbor, N.Y.
This work was supported by projects BIO2007-67821 and BIO2010-22146 (MICINN, Spain), S-SAL-0246/2006 (CAM, Spain), GR58/08 (Ref. 920640) from UCM and Research Networking Programme (06-RNP-132; European Science Foundation). CN is head of the Merck Sharp & Dohme special chair in genomics and proteomics. Thanks are due to all members of the UCM-PCM Genomic Unit for their collaboration in managing the collection of yeast mutants and A. Vázquez of the Centro de Citometría y Microscopía de Fluorescencia (UCM, Spain) for expert help in flow cytometry. We also thank members of Research Unit 4 at the Department of Microbiology for fruitful discussions, as well as María Molina and Humberto Martin for critical reading. Plasmids bearing SLT2/MPK1 alleles were kindly provided by Dr. D. Levin (Boston University).
PA was responsible for the assessment of levels of Slt2 phosphorylation, β-galactosidase assays, flow cytometry, phenotypic analysis and fluorescence microscopy. SD-M conducted the setting-up and subsequent screening for nourseothricin resistance with the collection of yeast mutants, and participated in fluorescence microscopy assays. RG carried out bioinformatic analysis and figure design. CN participated in the coordination of the study. JMR-P and JA conceived the study, participated in the design and analysis of experimental data and wrote the manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1:Complete set of yeast mutants with increased levels of Slt2 phosphorylation. Representative Western blot experiments of yeast mutants in which MAPK Slt2 was constitutively activated are shown. (PDF 311 KB)
Additional file 2:Yeast mutant strains resistant to nourseothricin without increased levels of Phospho-Slt2. Functional information on the genes whose disruption leads to antibiotic resistance without detectable Slt2 phosphorylation is shown. (PDF 10 KB)
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Arias, P., Díez-Muñiz, S., García, R. et al. Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: Novel insights into diverse MAPK outcomes. BMC Genomics 12, 390 (2011). https://doi.org/10.1186/1471-2164-12-390
- Endoplasmic Reticulum Stress
- Unfolded Protein Response
- Cell Wall Integrity
- Chitin Content