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Eukaryot Cell. Mar 2006; 5(3): 507–517.
PMCID: PMC1398071

The RIM101 Pathway Contributes to Yeast Cell Wall Assembly and Its Function Becomes Essential in the Absence of Mitogen-Activated Protein Kinase Slt2p

Abstract

The Saccharomyces cerevisiae ynl294cΔ (rim21Δ) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Δ also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Δ and rim101Δ mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Δ, as well as rim101Δ, was synthetic lethal with slt2Δ, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Δ rlm1Δ and rim101Δ rlm1Δ double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.

Cell walls surround yeast cells acting as an exoskeleton that confers cell shape, while protecting them from harsh environments (8). They are formed by different types of molecules, including structural components, such as glucans and chitin, as well as other molecules, such as different types of mannoproteins. These components are interconnected, although the nature of such interconnections is only partially known (5, 40). Despite the wealth of information collected about yeast cell walls, the specific role of many of the cell-wall-associated proteins remains unknown.

The importance of the cell wall in yeast biology is highlighted by the fact that inhibition of the synthesis of any of its structural components leads to cell death, making the yeast cell wall an attractive target for antifungal therapy (45). Yeast cells contain a dedicated mechanism, the cell integrity or protein kinase C (PKC) signal transduction pathway, which allows the cells to respond to cell wall damage (24). The response signal is executed by means of the transcriptional induction of several genes, mainly mediated by the Rlm1p transcription factor (13, 21). This transcriptional response is known as the compensatory response since it compensates for the damage produced in cell walls. Cell wall damage is sensed by a functional family of plasma membrane proteins whose two most important members are Wsc1p and Mid2p (19, 32, 44). Although their exact roles are not fully understood, they are partially redundant, and the simultaneous absence of both is lethal (16). After damage has occurred, the signal is transmitted through a kinase cascade whose distal mitogen-activated protein (MAP) kinase is Slt2p, which in turn phosphorylates Rlm1p, leading to its activation (for a review, see reference 24). According to its biological function, the PKC route is also constitutively induced in most mutants affected in cell wall structure (21). However, the PKC response is also induced by other types of stressful conditions, such as heat shock or caffeine treatment, whose effects cannot be directly related to cell wall structure (10, 27). Besides its role as a defense mechanism, the PKC cascade is also involved in the coordination of cell wall expansion during the cell cycle. In view of its different cellular roles, it is not surprising that mutations in the different components of the cascade lead to diverse phenotypes, which have allowed the identification of several cellular targets specific for each component of the cascade. Such targets have recently been reviewed (24) and have been grouped into two very different cellular categories: nuclear and cytoplasmic. At present, only a few targets for Slt2p MAP kinase have been established, including two transcription factors, a protein phosphatase, and a cell surface Ca2+ channel. However, genetic evidence has implicated Slt2p in the control of several additional hitherto-undefined substrates (24).

Although the role of PKC signaling in cell wall synthesis and assembly is well established, recent advances in our knowledge of signaling transduction processes strongly suggest that most, if not all, yeast signaling pathways also modulate this process. The HOG route (18) acts antagonistically to PKC in the construction of the yeast cell wall (14) and the compensatory response depends partially on the induction of the transcription factor Crz1p by the calcineurin pathway (13, 21). In addition, it has been known for some time that the sporulation signaling cascade directs spore cell wall synthesis in a timely fashion (7, 20) and that proper mating depends on the PKC response (33). Such relationships are not unexpected since the cell wall is in direct contact with the environment and therefore constitutes a major barrier against any environmental change.

In addition to the signaling pathways described above, fungi, including yeasts, contain a dedicated signal transduction pathway that is activated at alkaline pH: the so-called RIM101 pathway in Saccharomyces cerevisiae (31). RIM101 signaling, as well as all fungal alkaline pH systems, is atypical since it is not based on a MAP kinase cascade but rather on proteolytic processing of a transcription factor (30, 31), known as Rim101p in S. cerevisiae (25). Although the RIM101 pathway was described in S. cerevisiae several years ago in the context of its involvement in sporulation (28), most of our knowledge about this route comes from studies on filamentous fungi or Candida albicans. The signaling machinery appears to be evolutionarily conserved in most fungi, and the most recent hypothesis suggests that the transcription factor is proteolytically processed at the late-endosome membranes (for a review, see reference 30). Nevertheless, how the sensors of this route transmit the signal remains unknown. In S. cerevisiae the RIM cascade is formed by the products of the RIM8 (25), RIM9 (9), RIM13 (12), RIM20 (47), RIM21 (2, 42), and DFG16 (2) genes in addition to the transcription factor Rim101p (25), although the function of some of them is not well established.

The yeast transcriptional response mediated by Rim101p has been characterized by using DNA array-based technology (22, 23, 39) and has provided some unexpected answers: Rim101p functions as a repressor in vivo by downregulating the expression of several genes, including the transcription factors NRG1 and SMP1 (22), which themselves act as transcriptional repressors. Therefore, the absence of Rim101p produces both an increase and a decrease in the expression of different genes. How Rim101p exerts its function is not completely understood, and the regulatory response differs between S. cerevisiae (22) and C. albicans (26).

Direct experimental evidence linking the RIM101 signaling pathway and cell wall construction in yeast has not yet been obtained, but a very recent report indicates that some rim mutants show synthetic lethality with some cell wall mutations (41). In addition, some cell-wall-affected mutants display reduced growth at alkaline pH (38). Both observations, although not conclusive, suggest a possible relationship between the RIM101 pathway and S. cerevisiae cell wall assembly. More importantly, the expression of PHR1 and PHR2, as well as that of many other cell-wall-related genes, are pH regulated by the RIM101 pathway in C. albicans (26). CaPhr1/2p are the functional homologues of ScGas1p and Aspergillus fumigatus Gel1p proteins, two well-characterized glycosyl-transferase enzymes with essential functions in the construction of fungal cell walls (29). Unfortunately, the fact that the GAS1 gene is not under the control of the RIM101 pathway either in S. cerevisiae or in A. fumigatus does not support the relationship between RIM101 and cell wall assembly.

We present here direct experimental evidence linking the RIM101 and PKC pathways in the construction of S. cerevisiae cell walls. Both routes appear to act in parallel in the process, and the genetic interactions between them could open new alternatives for the therapeutic inhibition of fungal growth.

MATERIALS AND METHODS

Strains, plasmids, and yeast genetic methods.

Standard procedures were used for yeast genetic manipulations (35) and DNA manipulations (36). Plasmid pWL86, a pRS314 vector containing a RIM101 gene version truncated at the codon 531 (Rim101-531p), was kindly provided by A. P. Mitchell (25). The S. cerevisiae strains used in the present study and their origins are listed in Table Table1.1. Single mutants were made by the one-step gene replacement technique, while double mutants were obtained after tetrad dissection of the double heterozygous mutants obtained after conjugation of the appropriate single mutant strains.

TABLE 1.
Yeast strains

Single kanMX4 mutants were obtained from EUROSCARF, and the deletion cassettes were amplified from the genomic DNA of these strains by PCR with primers placed 200 nucleotides up- and downstream from each open reading frame. The mid2::kanMX4 deletion cassette was kindly provided by H. Bussey. The deletion cassettes were transformed into the appropriate strains, and correct transformants were verified by PCR and phenotypic analysis. The chs3::URA3, fks1::URA3, pbs2::LEU2, rlm1::LEU2, and slt2::URA3 deletion cassettes have been described elsewhere.

The rim21::URA3 deletion cassette was constructed as follows. P1 and P2 DNA fragments about 250 bp up- and downstream from the RIM21 open reading frame, respectively, were PCR amplified by using oligonucleotides 5′-GTACTACAGATTCGGTGTCC-3′ and 5′-CTAAGCTTCTGCATATACTGCCG-3′ (P1) and oligonucleotides 5′-TCAAGCTTCCAAACAGAGAAAGGCCA-3′ and 5′-TATGGCCTAGGTCGC-3′ (P2). Both fragments were cloned into the pGEMT vector (Promega), and the resulting plasmid was linearized with HindIII, where a HindIII-fragment containing the URA3 was inserted between the P1 and P2 fragments (pFMC1). The fragment containing the deletion cassette was transformed into different strains as described above to obtain the rim21Δ::URA3 strains.

Culture conditions.

YEPD (1% yeast extract, 2% peptone, and 2% glucose) broth or agar was the complete medium used for growing yeast strains. Synthetic minimal medium (SC; 0. 7% yeast nitrogen base without amino acids and 2% glucose) was routinely used after supplementation with appropriate amino acids and nucleic acid bases.

Growth in the presence of NaCl, sorbitol, caffeine, and sodium dodecyl sulfate (SDS) was always carried out on YEPD medium supplemented with different concentrations of the compounds, while resistance to calcofluor was assayed on SC medium buffered with 50 mM potassium hydrogen phathalate (pH 6.2). Sensitivity to alkaline pH was tested on YEPD medium, buffered with 100 mM HEPES adjusted with NaOH to the desired pH. For sensitivity tests, cells were grown overnight in the appropriate medium and diluted to an optical density at 600 nm (OD600) of approximately 0.2 to 0.3. Then, 5 μl of 1/10 serial diluted samples were spotted onto the indicated media. Growth was recorded after 48 h of incubation at 28°C.

The calcofluor effect was also tested in cells grown for 3 h in liquid YEPD supplemented with 0.075 mg of calcofluor/ml. The effect of this drug was tested by observation of septa enlargement generated under a Leica DRM500 fluorescence microscope or by the determination of the cell wall chitin levels. Fluorescence images were acquired with a Sensys digital camera and further processed with the Adobe Photoshop 5.5 software. All images were exposed and processed identically for accurate analysis.

Preparation of yeast extracts and immunoblot analyses.

Yeast cells were grown overnight at 24°C to mid-log phase in the appropriate medium, diluted to an OD600 of 0.2, and grown for 1 h. Then, cells were treated with the required compound or shifted to 39°C for the indicated times. Cells were collected on ice-chilled water in a refrigerated centrifuge, transferred to a microcentrifuge tube, immediately pelleted, and frozen in dry ice. Cells were lysed in 120 μl of cold lysis buffer (50 mM Tris-HCI [pH 8], 1% Triton X-100, 150 mM NaCl, 10 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and the protease inhibitors TPCK [tolylsulfonyl phenylalanyl chloromethyl ketone], tosyllysine chloromethyl ketone, leupeptin, pepstatin A, antipain, and aprotinin [each at 25 μg/ml]) by vigorous shaking with 0.45-mm glass beads in a Fast Prep cell breaker (Bio 101). Cell extracts were separated from the glass beads and cell debris and further clarified by a 16,000 × g spin for 15 min at 4°C. The protein concentration of the extract was determined at 280 nm, and samples were diluted in 2× loading buffer. Samples containing 100 mg of proteins were boiled for 5 min and fractionated in 10% SDS-polyacrylamide gels.

Western blots were carried out as described previously (27) by using an anti-phospho-p44/42 MAP kinase antibody (New England Biolabs) at a 1:500 dilution or an anti-Mpk1p (yN-19) goat polyclonal antibody to detect dually phosphorylated Slt2p or total Slt2p, respectively. Blots were developed by using the corresponding horseradish peroxidase-conjugated secondary antibody together with the ECL (Amersham Biosciences) detection system.

Zymolyase sensitivity assays.

Yeast sensitivity to zymolyase was assayed as growth inhibition, as described previously (1). Briefly, cells were pregrown, diluted to an OD600 of 0.005, and further incubated for 24 h at 28°C in YEPD or 1 M sorbitol-supplemented YEPD in the presence of different Zymolyase 100T (Seikagaku Corp.) concentrations. Growth was then determined by measurement of the OD600 of each culture. The data are expressed as the percentage of growth relative to the culture grown without zymolyase.

Chitin determinations.

Chitin measurements were performed with chitinase from Serratia marcescens (Sigma-Aldrich) and colorimetric determination of GlcNAc as described previously (43). Total amounts of chitin are expressed as millimoles of GlcNAc in 100 mg of cells (wet weight).

Electron microscopy.

Transmission electron microscopy (TEM) was performed essentially as described previously (46). Briefly, S. cerevisiae cultures were grown in 1 M sorbitol-supplemented YEPD to an OD600 of 0.5 to 1.0. Then, 12.5 OD600 units of cells were collected by centrifugation, rapidly washed, and fixed in 2% potassium permanganate for 1 h at room temperature. Excess potassium permanganate was removed by exhaustive washing, and the stained cells were finally dehydrated by incubation in increasing concentrations of ethanol. Samples were processed in the embedding medium (Spurr Resin Embedding Kit; TAAB) by successive 2-h incubations in 1:1 and 1:3 ethanol-embedding medium mixtures and, finally, in fresh embedding medium. Cells were concentrated in 500 μl of resin, and samples were allowed to polymerize overnight at 60°C. Ultrathin sections were obtained by using an LKB Ultratome III microtome, and samples were visualized under a Zeiss EM900 transmission electron microscope. Images were processed with the Adobe Photoshop software, preserving relative magnifications. Cell wall widths were determined in several cells of each strain, and the individual value for each cell is the average of eight different measurements along its surface.

RESULTS

Identification of YNL294 as a cell wall-related gene.

The participation of our laboratory in the EUROFAN project allowed us the possibility of screening multiple nonessential mutants for cell-wall-associated phenotypes. Among them, the ynl294Δ mutant showed calcofluor resistance and sensitivity to zymolyase, two phenotypic traits also associated with the lack of CSIII activity (17). However, ynl294Δ appeared to contain normal amounts of CSIII, which prompted us to investigate the reason for the calcofluor resistance in this mutant.

During the course of the present study the Ynl294 protein was shown to have modest but significant identity with A. nidulans PalHp (42). The functional characterization of this gene placed it in the ambient pH signaling pathway, known as the RIM101 pathway in S. cerevisiae. This gene was named RIM21 in all major databases, and it has been assumed to be the functional homologue of A. nidulans PalHp. Therefore, we shall refer to it as RIM21/Rim21p throughout the present study.

rim21Δ cells were resistant to moderate concentrations of calcofluor (Fig. (Fig.1A)1A) and, in accordance with the reported CSIII level (17), they contained similar amounts of chitin as the wild type (Table (Table2).2). After calcofluor treatment, chitin was localized at the septum region as in the wild type, but the intensity of the staining was significantly reduced (Fig. (Fig.1B),1B), a clear indication that chitin levels should be reduced in these conditions. This turned out to be the case, as rim21Δ cells contained only 44% of the chitin contained in wild-type cells after calcofluor treatment (Table (Table2).2). This reduction was due to the lower increase in chitin biosynthesis occurring in the mutant after calcofluor treatment. Interestingly, this situation was very similar to that observed for the slt2Δ and rlm1Δ mutants in terms of chitin synthesis (Table (Table2)2) or calcofluor staining (not shown). Since both slt2Δ and rlm1Δ mutants are defective in the cell integrity signaling (PKC) response, it was necessary to address the phenotypic traits associated with the absence of this route.

FIG. 1.
Phenotypic characterization of the rim21Δ mutant. (A) Resistance to calcofluor on SC medium. Plates were incubated for 48 h at 28°C. (B) Chitin distribution after calcofluor (0.075 mg/ml) treatment for 3 h. Note the lower intensity of ...
TABLE 2.
Chitin levels in selected strains

rim21Δ growth was significantly reduced at pH above 7.6 (Fig. (Fig.1C).1C). In addition, the mutant grew poorly in 0.9 M NaCl (Fig. (Fig.1C),1C), but no significant defect in sporulation was observed. These phenotypes clearly agree with the proposed model of Rim21p as part of the alkaline pH signaling response (see Discussion for further analysis).

rim21Δ growth was also reduced in the presence of 4 mM caffeine and 0.002% SDS (Fig. (Fig.1C),1C), but it was not affected by 1.5 M sorbitol (Fig. (Fig.1C)1C) or high temperature (not shown). Apparently, this mutant is moderately hypersensitive to SDS and caffeine but not to high osmolarity. These phenotypes have been traditionally associated with defects in the cell wall structure.

The rim21Δ mutant fails to activate the PKC response after cell wall damage.

The results obtained showed that, although to a different extent, the rim21Δ and slt2Δ mutants shared multiple phenotypes, pointing to Rim21p as a part of the cell integrity signaling response (PKC). In order to confirm this point, we determined the degree of the PKC response in the rim21Δ mutant after calcofluor treatment. In the wild-type cells, calcofluor induced a rapid and long-lasting phosphorylation of Slt2p in response to the cell wall damage produced (Fig. (Fig.2A).2A). In contrast, Slt2p phosphorylation was virtually absent in the rim21Δ mutant (Fig. (Fig.2A)2A) under similar growth conditions. The absence of this response is not a consequence of secondary mutations in this strain since it is also absent in an independent deletion strain (FMC28) and in the original FY1679 Δynl294::kanMX4 mutant (data not shown). In conclusion, deletion of RIM21 abolished PKC response after calcofluor treatment. The fks1Δ mutant has an altered cell wall synthesis, and hence it shows a constitutive activation of the PKC pathway as determined by the degree of Slt2p phosphorylation (Fig. (Fig.2B)2B) and the increase in chitin synthesis (Table (Table2).2). However, Slt2p phosphorylation was not observed in the double fks1Δ rim21Δ mutant and thus chitin synthesis was reduced (Table (Table2).2). Taken together, these results indicated that the PKC response was not elicited in the rim21Δ mutant after cell wall damage produced by two completely different mechanisms. In contrast, Slt2p phosphorylation was induced in the rim21Δ mutant by incubation at high temperatures or after caffeine treatment (Fig. (Fig.2C),2C), another two stressful situations in which the PKC response is activated (Fig. (Fig.2C)2C) (27). Apparently, Rim21p is specifically involved in the activation of the PKC response after direct cell wall damage.

FIG. 2.
Activation of the PKC signaling pathway under different conditions. (A) Time course of Slt2p phosphorylation after calcofluor treatment. Samples were collected at the indicated times after the addition of calcofluor. The levels of total Slt2p are shown ...

In order to further confirm the relationship between Rim21p and cell wall construction, we analyzed the hypothetical synthetic lethality between rim21Δ and several other mutants previously shown to have altered cell walls. chs3Δ rim21Δ, pbs2Δ rim21Δ, fks1Δ rim21Δ, mid2Δ rim21Δ, and wsc1Δ rim21Δ double mutants were viable and did not show any apparent defect under normal growth conditions (data not shown). However, we were unable to obtain the double slt2Δ rim21Δ mutant, suggesting a synthetic lethal interaction between both mutations (see below). Further characterization of the double mutants indicated that the phenotypic defects detected were additive (Fig. (Fig.33 and data not shown). However, the fks1Δ rim21Δ mutant showed an extreme hypersensitivity to SDS (Fig. (Fig.3)3) that could not be explained by the additive effect of both mutations.

FIG. 3.
Phenotypic analysis of selected double mutants. Early logarithmic growing cells were spotted onto the indicated media at 1/10 serial dilutions. Growth was scored after 48 h at 28°C. Note the extreme hypersensitivity to SDS of the double fks1Δ ...

We have previously stated that slt2Δ rim21Δ double mutants appeared to be nonviable. In order to confirm this lethality, we performed a statistical analysis of rim21Δ and slt2Δ segregation. As expected, no slt2Δ rim21Δ double mutants were obtained after the micromanipulation of 34 tetrads (Table (Table3),3), albeit the normal segregation of the two characters occurred individually, a clear indication of the synthetic lethality between both mutations. In order to analyze whether or not such lethality might be directly related to a cell wall defect, we performed the segregation analysis on plates supplemented with 1 M sorbitol. As shown in Table Table3,3, from the spores analyzed we were able to recover a significant number of double mutants, which accounted for ca. 60% of those expected based on the appearance of single mutations. Apparently, rim21Δ slt2Δ synthetic lethality can be suppressed by osmotic stabilization of the growth media, suggesting a severe defect in cell wall assembly in this double mutant.

TABLE 3.
Spore viabilitya in different crosses

The RIM pathway is involved in cell wall construction.

All of the results presented thus far link the phenotypes of the rim21Δ mutation to the construction of the yeast cell wall through the participation of Rim21p in the cell integrity signaling response. In addition, according to its structure and functional similarity to AnPalHp (2, 42), Rim21p appears to be a membrane protein that acts as a sensor. We were therefore prompted to place Rim21p as an additional sensor of the PKC pathway. If this were the case, we reasoned that we would not find a relationship between RIM101 and PKC pathways but rather an involvement of Rim21p in both routes. In order to test this point, we characterized the cell-wall-associated phenotypes of the rim101Δ mutant, which lacks the transcription factor associated with the RIM101 response.

The rim101Δ mutant was partially resistant to calcofluor and hypersensitive to SDS and caffeine (results not shown). It contained normal amounts of chitin, but calcofluor treatment increased chitin synthesis only by a factor of 2 (Table (Table2),2), in a way similar to what occurred for the rim21Δ mutant. In addition, we were unable to obtain the double rim101Δ slt2Δ mutant in the W303 genetic background (Table (Table3),3), a clear indication of the synthetic lethality between both mutations. This lethality could also be partially overcome by osmotic stabilization of the media (Table (Table3).3). The similarity between rim21Δ and rim101Δ phenotypes strongly supports the relationship between the RIM101 and PKC routes rather than a direct role of Rim21p in the PKC cascade. In order to confirm this hypothesis, we analyzed the complementation behavior of a plasmid containing the C-terminal deletion of Rim101p which produces its constitutive activation (25) and hence induction of the RIM pathway. The introduction of plasmid pWL86 (Rim101-531p) in the rim21Δ mutant restored alkaline pH growth (not shown), calcofluor sensitivity (Fig. (Fig.4A),4A), and the PKC response after calcofluor treatment (Fig. (Fig.4B),4B), without having a direct effect per se on the induction of the PKC response (Fig. (Fig.4B).4B). In clear accordance with these results, Rim101-531p also promoted the growth of the otherwise lethal rim21Δ slt2Δ mutant. However, the suppression of this lethality was only partial and quantitatively very similar to that obtained by osmotic stabilization (Table (Table3).3). This experiment also allowed us to reconfirm the lethality of the slt2Δ rim21Δ double mutant. It was able to grow containing the pWL86 plasmid, but we were unable to induce plasmid shuffling in this strain even after 30 generations of growth in nonselective media, where other strains lose plasmids with a frequency close to 50% (Table (Table33).

FIG. 4.
Effect of the expression of the constitutive form of Rim101p. Wild-type and rim21Δ strains were transformed with pWL86 (Rim101-531p) and characterized phenotypically. (A) Resistance to calcofluor on SC medium. (B) Slt2p phosphorylation before ...

In conclusion, simultaneous inactivation of the RIM101 and PKC signaling pathways leads to nonviable cells. However, it should be noticed that neither the rim21Δ or rim101Δ mutations showed synthetic lethality with the rlm1Δ mutant (Table (Table3)3) and that the double mutants grew normally. This mutant lacks the only transcription factor that has been experimentally implicated in the PKC-induced transcriptional response, thus pointing to additional levels of control of cell wall assembly through the PKC route.

Deletion of the Smp1p transcriptional repressor has minor effects on cell wall-associated rim21Δ defects.

Recent advances in the regulatory network of RIM101 signaling in S. cerevisiae have pointed to an important role of two transcriptional repressors, Nrg1p and Smp1p, in the process (22). Although there is no experimental evidence implicating either of them in cell wall assembly (http://www.yeastgenome.org/), Smp1p is the only known homologue of the Rlm1p transcription factor in S. cerevisiae (11). It was therefore tempting to associate the role of RIM signaling in cell wall assembly with a Smp1p deregulation. In order to test this possibility, we characterized the smp1Δ and smp1Δ rim21Δ phenotypes. The smp1Δ strains did not show any cell-wall-associated phenotype (Fig. (Fig.5)5) but, more interestingly, deletion of the SMP1 gene did not suppress the hypersensitivity of a rim21Δ mutant to SDS or NaCl (Fig. (Fig.5B).5B). Double mutants were still moderately resistant to calcofluor (Fig. (Fig.5B),5B), and the effect of this drug was lower than in the wild type, as determined by calcofluor staining (Fig. (Fig.5A).5A). However, its resistance to calcofluor seemed to be lower than that of the single rim21Δ. In addition, after calcofluor treatment the amount of chitin was significantly higher than in rim21Δ but still lower than in the wild type (Fig. (Fig.5A).5A). These results clearly ruled out the derepression of SMP1 as the only factor linking the RIM101 pathway with cell wall assembly, suggesting a more complex mechanism in which derepression of SMP1 could be partially involved.

FIG. 5.
Phenotypic characterization of the smp1Δ mutants. (A) Calcofluor staining of the indicated mutants and chitin levels after calcofluor treatment measured as described in Materials and Methods. In parentheses, the relative chitin levels are indicated ...

Double RIM and PKC mutants can only grow in osmotically stabilized media because of severe cell wall defects.

In a further attempt to understand the role of the RIM101 pathway in cell wall construction, we analyzed the behavior of the double mutants isolated. As previously stated, slt2Δ rim21Δ or slt2Δ rim101Δ double mutants were only isolated on YEPD media supplemented with 1 M sorbitol. When these strains were transferred to plain YEPD media, growth stopped and almost no growth was detected on solid (Fig. (Fig.6A)6A) or liquid (not shown) medium. Although growth on sorbitol-supplemented solid medium did not indicate any apparent growth defect of slt2Δ rim21Δ/101 double mutants, after inoculation of cultures in liquid media slt2Δ rim21Δ growth was significantly slower (Fig. (Fig.6B).6B). The generation time of the double mutant was 222 min, approximately twice as long as any of the single rim21Δ or slt2Δ mutants, with 112 and 110 min, respectively.

FIG. 6.
Growth of the indicated strains in different media. (A) Complete tetrads grown on YEPD-1 M sorbitol were transferred in parallel to YEPD and YEPD-1 M sorbitol and incubated for 3 days at 28°C. Note the minor growth of the double mutants on plain ...

The synthetic lethality of the slt2Δ rim21Δ/101 mutants was strain dependent since these double mutants in the FY1016 genetic background were able to grow on plain YEPD. However, growth was very slow at 28°C, and the strain was essentially thermosensitive at 37°C (not shown), confirming the synthetic interaction between the RIM101 and PKC pathways in two different genetic backgrounds.

In order to discover whether this genetic interaction was related to cell-wall-associated defects, as expected from phenotypic characterization of the individual mutants, we carried out a sensitivity test to zymolyase, an enzymatic cocktail that acts on fungal cell walls (see Materials and Methods for the specific protocol). In YEPD medium, slt2Δ cells were extremely hypersensitive to zymolyase, and these cells did not grow when zymolyase concentrations were as low as 25 U (Fig. (Fig.7A).7A). pbs2Δ cells, used as control, showed a moderate hypersensitivity to zymolyase. The rim21Δ and rim101Δ single mutants showed a modest but reproducible hypersensitivity to the enzyme since their growth was reduced in the presence of zymolyase, although they were still able to grow substantially even at the highest concentration used (Fig. (Fig.7A).7A). Characterization of the double rim101Δ slt2Δ mutant was necessarily carried out on YEPD with 1 M sorbitol. In this medium, resistance to zymolyase was significantly improved in all of the strains tested and the slt2Δ single mutant became as resistant to zymolyase as the wild type (Fig. (Fig.7B).7B). Interestingly, rim101Δ growth was increased reproducibly at low concentrations of zymolyase (Fig. 7B and C). However, the double rim101Δ slt2Δ mutant was very hypersensitive to zymolyase compared to the single mutants (Fig. (Fig.7B).7B). Similar results were obtained for the rim21Δ slt2Δ mutant (data not shown).

FIG. 7.
Sensitivity of different strains to zymolyase. (A) Growth of indicated strains in increasing concentrations of zymolyase in YEPD media. The data are expressed as percentages of growth compared to identical cultures grown without zymolyase. See Materials ...

Further characterization of these mutants was performed by TEM of cells grown in 1 M sorbitol. The overall aspect of the double mutant cells was similar to those of the single mutants and the controls (Fig. (Fig.8A),8A), but closer scrutiny (Fig. (Fig.8B)8B) revealed some differences in the double mutant. The rim21Δ slt2Δ cell walls were 0.217 ± 0.006 μm in width, significantly (50%) wider than those of the wild-type (0.149 ± 0.01 μm), rim21Δ (0.143 ± 0.009 μm), or slt2Δ (0.136 ± 0.008 μm) cells. In addition, the darker layers flanking the inner core of the cell wall appeared less defined, suggesting an altered assembly of the cell wall structure.

FIG. 8.
TEM images of the indicated strains grown in YEPD-1 M sorbitol. Cells are presented at two different magnification levels to show details. Note the less-defined outer layer, corresponding to the mannoprotein layer (B). WT, wild type. Bars, 0.4 μm. ...

DISCUSSION

Ynl294/Rim21p forms a functional part of the alkaline pH signaling pathway but also participates in cell wall construction.

Yeast cells respond to damage to their cell walls almost exclusively through the induction of the PKC pathway, which triggers a transcriptional response mediated by the Rlm1p transcription factor (13, 21). However, recent advances in our knowledge of the mechanisms of cell wall synthesis and assembly point to multiple inputs to the regulation of this process (for a review, see reference 24). Therefore, it is not surprising to find multiple mutations conferring cell-wall-associated defects through the use of massive screenings. In one of these screenings, partially carried out in our lab, the resistance of the ynl294Δ mutant to calcofluor was uncovered (17). However, this mutant contained normal levels of CSIII. Previously, this combination of phenotypes had been only found in different mutants of the HOG pathway (14). It was therefore tempting to associate Ynl294p with a signaling cascade involved in cell wall assembly.

During the course of this work, Ynl294p was named Rim21p as part of the alkaline pH signaling response (RIM101) in S. cerevisiae (2, 42). Our results confirmed this participation since rim21Δ mutants grew poorly at pH 7.6 and showed a significant hypersensitivity to NaCl (Fig. (Fig.1),1), both phenotypes expected from the alteration of the regulatory network associated with the RIM101 response. In addition, the Δrim21 mutant also showed a strong defect in mating (data not shown), whose characterization will be presented elsewhere. Interestingly, our results also highlight the cell-wall-associated defects displayed by the rim21Δ mutant. Therefore, our main goal was to know whether Rim21p, or alternatively the RIM101 pathway, was involved in cell wall assembly.

The S. cerevisiae RIM101 pathway acts on cell wall structure.

The many cell-wall-associated phenotypes of the rim mutants described here can be simply explained in terms of the absence of a functional PKC response after cell wall damage (Fig. 2A and B), even though PKC is activated by other stimuli (Fig. (Fig.2C).2C). However, if this model is correct then the rim21Δ mutant should have a synthetic lethality pattern similar to that of the slt2Δ mutant, which is not the case: chs3Δ rim21Δ or fks1Δ rim21Δ mutants were viable and, more importantly, the slt2Δ rim21Δ double mutant turned out to be lethal. The synthetic lethality was relieved by osmotic stabilization of the media, suggesting that Rim21p and Slt2p act in parallel in the construction of the yeast cell wall. These results also argue against a role of Rim21p as a sensor in the PKC cascade, although is still possible that it could be involved in the process as part of another signaling cascade, as could be speculated from its biological role as a PM sensor (2, 42).

In order to test this possibility, we refocused our attention on Rim101p, the transcription factor involved in the RIM101 response. rim101Δ cells showed essentially the same phenotypes as rim21Δ cells. In addition, the rim101Δ and slt2Δ mutations were also synthetically lethal. More important, the phenotypes shown by the rim21Δ mutant were reversed by the expression of the constitutively activated form of Rim101p (25) (Fig. (Fig.4).4). This reversal included not only growth at alkaline pH but also calcofluor sensitivity, activation of the PKC response after cell wall damage, and synthetic lethality with the slt2Δ mutation. These results allow us to confirm that the RIM101 pathway, and not only Rim21p, acts on the formation of yeast cell wall. This relationship has also been recently uncovered in massive synthetic lethality analyses which show that slt2Δ displays synthetic lethality with the rim20Δ, rim13Δ, and rim8Δ mutations (41). Surprisingly, neither the rim101Δ nor the rim21Δ mutations showed synthetic lethality with the slt2Δ mutation in this screening, a finding very likely due to methodological problems associated with massive screenings.

Once the relationship between RIM101 and the cell wall has been established, the next step would be to determine the exact role of the RIM101 pathway in this process. However, this is very difficult, owing to the complex nature of the cell wall components and their interactions. The mild phenotypes found for the Δrim mutants, as well as their modest hypersensitivity to zymolyase, suggested minor changes in cell wall structure. These changes very likely affect the cell wall interaction with calcofluor, leading to drug resistance and the virtual absence of the PKC response. These minor defects became lethal in the absence of slt2Δ, but not in the rlm1Δ mutant, which lacks the transcriptional response mediated by PKC activation after cell wall damage (13, 21). These results strongly support a model in which the RIM and PKC pathways act in parallel in a group of complementary functions required for proper cell wall assembly. The specific functions of these genes are very hard to define because Slt2p appears to have multiple intracellular targets not yet defined (24). One such target is the Ca2+ channel Cch1p/Mid2p (4). However, the genetic interaction described here appears to be independent of Ca2+ signaling since the absence of the calcineurin proteins was not synthetic lethal with rim21Δ and neither did overexpression of the constitutively active calcineurin regulatory subunit (15) suppress the synthetic lethality of the rim21Δ slt2Δ mutant (data not shown).

Unfortunately, transcriptional insight into the RIM101 pathway is also very complex. The yeast RIM101 pathway has been implicated in transcriptional repression (3, 22) and transcriptional induction (3, 23, 39), but it is also required for the expression of several genes (22). The three categories include genes that potentially participate in the construction of yeast cell walls and hence the three regulatory mechanisms could interact in the production of the phenotypes described. In agreement with this hypothesis, deletion of the transcriptional repressor smp1Δ only partially suppressed the cell wall-associated phenotypes of the rim21Δ mutant. The massive synthetic lethality screening recently reported (41) also argues against a single gene being responsible for the phenomena discussed here since none of the multiple genes regulated by the RIM101 pathway showed synthetic lethality with slt2Δ except for the RIM genes.

Interestingly, several of the genes regulated by the RIM101 pathway in S. cerevisiae code for cell wall remodeling activities, such as CRH1, CRH2 (34), or CTS1 (6). It is thus very likely that a combinatorial effect of several remodeling activities would account for the phenotypes found in the rimΔ mutants. This hypothetical defect in cell wall remodeling rather than in structural activities would provide a more suitable explanation for the mild structural defects observed in the cell walls of the double mutants.

The functional relationship between the RIM101 and PKC routes is also becoming apparent from other evidence. The C. albicans PHR1 and PHR2 genes are regulated by pH (37) and, like other fungal homologues such as the S. cerevisiae GAS1 and the A. fumigatus GEL1, they play essential roles in cell wall construction (29, 37). Although there is no evidence about pH regulation of the GAS1 or GEL1 genes, the gas1Δ and the slt2Δ and bck1Δ mutants have recently been shown to display a moderate hypersensitivity to alkaline pH (38). Therefore, yeast cells appear to require cell wall remodeling for growth at high pH. If this hypothesis is correct, then very likely alkaline pH would trigger the PKC response. This is the case since the growth of S. cerevisiae at pH 7.6 induced a transient phosphorylation of Slt2p depending on the presence of Rim21p (data not shown). In conclusion, the RIM101 and PKC pathways cooperate in cell wall remodeling after alkaline shock.

The main conclusion from the present study is that this is the first direct experimental evidence assigning a role to the RIM101 pathway in the assembly of the S. cerevisiae cell walls. This relationship should help to define the additional intracellular targets of Slt2p that have remained elusive thus far. To date, virtually all of the signal transduction pathways described in yeasts act on cell wall assembly, highlighting the dynamic nature of this structure that allows cellular adaptation in response to many different types of environmental changes.

Acknowledgments

We thank the members of the laboratory of A. Duran for critical comments on the manuscript and N. Skinner for language revision. Special thanks are due to A. P. Mitchell and M. S. Cyert for strains and plasmids and to all the members of the Eurocellwall project for useful comments.

M.S. acknowledges the financial support from the CSIC through the I3P-BPG2003 program. This research was supported by CICYT grant BIO2004-00280 and EU grants QLK3-CT-2000-01537 and LSHB-CT-2004-511952.

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