Rim101 Pathway Research Articles

Identical domains of Yarrowia lipolytica Vps23 are required for both ESCRT and Rim pathways, but the latter needs an interaction between the Vps23 UEV domain and Rim8/PalF

A conserved pathway, called Rim or Pal, transduces the ambient pH signal in ascomycetous yeasts and fungi, respectively. This pathway requires most of the components of the endosomal sorting complex required for transport (ESCRT) pathway. In the yeast Yarrowia lipolytica, a functional analysis of the ESCRT-I subunit Vps23 was carried out by in-frame deletions of each of the conserved domains to test whether Vps23 functions in the Rim and ESCRT pathways could be separated. These two pathways were shown to necessitate both the coiled-coil domain and the C-terminal steadiness box. However, the central proline-rich region seems to be required for neither of them. Both pathways involve the N-terminal ubiquitin E2 variant (UEV) domain. Thus, identical domains of YlVps23 are required for both Rim and ESCRT pathways, but the UEV domain was shown to bind the arrestin-like protein Rim8/PalF in the Rim pathway, whereas it binds Vps27 in the ESCRT pathway. Vps23 is therefore required to link pH signalling and endocytosis.

Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors

In the human fungal pathogen Candida albicans, environmental pH has profound effects on morphogenesis and response to extracellular pH is clearly relevant to the pathogenicity of this fungus. Yeast cells have evolved a complex network of mechanisms in response to the environmental pH and they often require the integration of the Rim101 and calcineurin/Crz1 signaling pathways. Ca(2+) burst is a common cellular response when cells are exposed to environmental stresses; therefore, in this study, we asked whether it follows the same case under alkaline stress and whether this calcium change is regulated by Rim101p and Crz1p. We confirmed the calcium influx was activated by KOH stimuli using a flow cytometry-based method, but it was obviously abolished in cells lacking MID1 or CCH1. We also found that alkaline pH-induced activation of the PHO89 promoter was blocked without the same gene; moreover, the response was Crz1p- and Rim101p-dependent. Finally, we investigated the regulation role of Rim101p and Crz1p in calcium influx through MID1, CCH1 and YVC1 genes, which were all downregulated in rim101Δ/Δ and crz1Δ/Δ mutants. The important role of calcium influx in the alkaline stress response and its regulation suggested a potential integration effect of Rim101 and Crz1 signaling pathways in C. albicans.

Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape.

Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A2 (PLA2s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA2 in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA2 activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA2 activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-010-0533-8) contains supplementary material, which is available to authorized users.

TheRIM101Signal Transduction Pathway RegulatesCandida albicansVirulence during Experimental Keratomycosis

To examine the role of the fungal RIM101 signal transduction pathway in the pathogenesis of Candida albicans keratitis. C. albicans wild-type strain SC5314, prototrophic mutant control DAY185, and homozygous fungal mutants for the rim8, rim13, rim20, rim101, and phr1 genes were evaluated in vitro using proliferation and filamentation assays. Scarified corneas of BALB/c and C57BL/6J mice were topically inoculated and observed daily for keratitis severity. Corneal adaptation and pathogenicity were assessed ex vivo by maintaining infected porcine corneas for 3 days in an explantation culture system for histologic evaluation of hyphal penetration. All C. albicans strains had similar growth kinetics, and SC5314 and DAY185 demonstrated pH-induced filamentation. Fungal mutants had reduced hyphal formation at alkaline and neutral pH, but normal acidic assays ascertained that mutant strains did not have a generalized filamentation defect. SC5314 and DAY185 caused moderate to severe keratitis in mice, whereas fungal strains lacking constituents of the RIM101 pathway had significantly (P<0.05) attenuated severity in vivo. Three days after inoculation of porcine corneas, SC5314 and DAY185 produced hyphae that penetrated 28% and 25%, respectively, of the corneal thickness, and all five mutant strains showed significantly (P<0.05) less stromal penetration. The RIM101 signal transduction pathway plays an important role in the development of C. albicans keratitis. The fungal pathway intermediates Rim8p, Rim13p, Rim20p, and Rim101p and the downstream cell-wall protein Phr1p are pivotal in the process of corneal invasion by C. albicans.

Mutational Analysis of Candida albicans SNF7 Reveals Genetically Separable Rim101 and ESCRT Functions and Demonstrates Divergence in bro1-Domain Protein Interactions

The opportunistic pathogen Candida albicans can grow over a wide pH range, which is associated with its ability to colonize and infect distinct host niches. C. albicans growth in neutral-alkaline environments requires proteolytic activation of the transcription factor Rim101. Rim101 activation requires Snf7, a member of the endosomal sorting complex required for transport (ESCRT) pathway. We hypothesized that Snf7 has distinct functions in the Rim101 and ESCRT pathways, which we tested by alanine-scanning mutagenesis. While some snf7 alleles conferred no defects, we identified alleles with solely ESCRT-dependent, solely Rim101-dependent, or both Rim101- and ESCRT-dependent defects. Thus, Snf7 function in these two pathways is at least partially separable. Both Rim101- and ESCRT-dependent functions require Snf7 recruitment to the endosomal membrane and alleles that disrupted both pathways were found to localize normally, suggesting a downstream defect. Most alleles that conferred solely Rim101-dependent defects were still able to process Rim101 normally under steady-state conditions. However, these same strains did display a kinetic defect in Rim101 processing. Several alleles with solely Rim101-dependent defects mapped to the C-terminal end of Snf7. Further analyses suggested that these mutations disrupted interactions with bro-domain proteins, Rim20 and Bro1, in overlapping but slightly divergent Snf7 domains.

The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Delta) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae

Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

Interaction of Cryptococcus neoformans Rim101 and Protein Kinase A Regulates Capsule

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101Δ mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101Δ strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101Δ mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions.

Analysis of the dual function of the ESCRT-III protein Snf7 in endocytic trafficking and in gene expression

ESCRT (endosomal sorting complex required for transport)-III mediates the budding and scission of intralumenal vesicles into multivesicular endosomes in yeast. For the main ESCRT-III subunit Snf7, an additional role in activation of the transcription factor Rim101 (the 'Rim pathway') is now also firmly established. In the present study, we investigate how these two Snf7 functions are related to each other. By generating SNF7 mutations that severely affect endocytic trafficking, but leave the Rim pathway function intact, we show that the two functions of SNF7 can be separated genetically. We analysed in detail how the SNF7 mutations affect the interaction of Snf7 with its various binding partners. Although the interactions with proteins Rim13 and Rim20, necessary for the Rim-pathway-related functions, were not altered by the mutations, there was a strong effect on interactions with components of the ESCRT pathway. The interactions, as measured by co-immunoprecipitation, with the ESCRT-III subunits Vps20 and Vps24 were strongly increased by the mutations, whereas the interactions with proteins Vps4 and Bro1, acting downstream of ESCRT-III, were reduced. As Vps4 is required for disassembly of ESCRT-III these results suggest that ESCRT-III is more stable in our SNF7 mutants. In line with this notion, a higher fraction of mutant Snf7 protein was detected at the membrane. Upon a shift to alkaline pH, a stronger binding signal for virtually all interaction partners, except Vps4, was observed. This indicates that the ESCRT network at the endosomal membrane is more extensive under these conditions.

Exploring the Mode of Action of Antimicrobial Peptide MUC7 12-Mer by Fitness Profiling of Saccharomyces cerevisiae Genomewide Mutant Collection

The MUC7 12-mer (RKSYKCLHKRCR) is a cationic antimicrobial peptide derived from the human salivary mucin MUC7. To study its effect/mechanism of action on fungi, we performed a fitness screen of a tagged, diploid, homozygous gene deletion mutant pool of the yeast Saccharomyces cerevisiae grown in the presence of the MUC7 peptide. Forty-five strains exhibiting reduced fitness and 13 strains exhibiting increased fitness (sensitivity or resistance, respectively) were identified by hybridization intensities to tag arrays. The strongest fitness defects were observed with deletions in genes encoding elements of the RIM101 signaling pathway (regulating response to alkaline and neutral pH and other environmental conditions) and of the endosomal sorting complex required for transport (ESCRT; functioning mainly in protein sorting for degradation, but also required for activation of the RIM101 pathway). Other deletions identified as conferring fitness defect or gain are in genes associated with a variety of functions, including transcription regulation, protein trafficking, transport, metabolism, and others. The results of the pool fitness screen were validated by a set of mutant strains tested individually in the presence of the MUC7 12-mer. All tested RIM101-related deletion strains showing fitness defects confirmed their sensitivities. Taken together, the results led us to conclude that deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide by preventing activation of this pathway and that this stress response plays a major role in the protection of S. cerevisiae against damage inflicted by the MUC7 12-mer peptide.

BAR Domain Proteins Rvs161 and Rvs167 Contribute to Candida albicans Endocytosis, Morphogenesis, and Virulence

The Candida albicans plasma membrane plays critical roles in growth and virulence and as a target for antifungal drugs. Three C. albicans genes that encode Bin-Amphiphysin-Rvs homology domain proteins were mutated to define their roles in plasma membrane function. The deletion of RVS161 and RVS167, but not RVS162, caused strong defects. The rvs161Delta mutant was more defective in endocytosis and morphogenesis than rvs167Delta, but both were strongly defective in polarizing actin patches. Other plasma membrane constituents were still properly localized, including a filipin-stained domain at the hyphal tips. An analysis of growth under different in vitro conditions showed that the rvs161Delta and rvs167Delta mutants grew less invasively in agar and also suggested that they have defects in cell wall synthesis and Rim101 pathway signaling. These mutants were also more resistant to the antimicrobial peptide histatin 5 but showed essentially normal responses to the drugs caspofungin and amphotericin. Surprisingly, the rvs161Delta mutant was more sensitive to fluconazole, whereas the rvs167Delta mutant was more resistant, indicating that these mutations cause overlapping but distinct effects on cells. The rvs161Delta and rvs167Delta mutants both showed greatly reduced virulence in mice. However, the mutants were capable of growing to high levels in kidneys. Histological analyses of infected kidneys revealed that these rvsDelta mutants grew in a large fungal mass that was walled off by leukocytes, rather than forming disseminated microabscesses as seen for the wild type. The diminished virulence is likely due to a combination of the morphogenesis defects that reduce invasive growth and altered cell wall construction that exposes proinflammatory components to the host immune system.

The RIM101 pathway has a role inSaccharomyces cerevisiaeadaptive response and resistance to propionic acid and other weak acids

The physiological function of the Saccharomyces cerevisiae RIM101 signaling pathway is extended in this study beyond alkaline pH-induced responses. The transcription factor Rim101p is demonstrated to be required for maximal tolerance to weak acid-induced stress, at pH 4.0, but does not exert protection against low pH itself (range 4.5-2.5), when a strong acid is used as the acidulant. The Rim101p-dependent alterations of the yeast transcriptome following exposure to propionic acid stress (at pH 4.0) include genes of the previously described Rim101p regulon but also new target genes, in particular KNH1, involved in cell wall beta-1,6-glucan synthesis and the uncharacterized ORF YIL029c, both required for maximal propionic acid resistance. Clustering of the genes that provide resistance to propionic acid reveals the enrichment of those involved in protein catabolism through the multivesicular body pathway and in the homeostasis of internal pH and vacuolar function. The analysis of the network of interactions established among all the identified propionic acid resistance determinants shows an enrichment of interactions around the RIM101 gene and highlights the role of proteins involved in Rim101p proteolytic processing. RIM101 expression is shown to be required to counteract propionic acid-induced cytosolic acidification and for proper vacuolar acidification and cell wall structure, these having positive implications for a robust adaptive response and resistance to stress promoted by this food preservative.

Candida albicansferric reductaseFRP1is regulated by direct interaction with Rim101p transcription factor

The opportunistic pathogen Candida albicans develops different systems to acquire essential nutrients such as iron. The Rim101 pathway controls gene expression to adapt to different environments through transcription factor Rim101p. Ferric reductase genes are important for iron acquisition, but further work should be carried out to show about how ferric reductase genes are regulated. In this study we demonstrate that ferric reductase FRP1 expression is upregulated by alkaline pH and iron-limited conditions. Moreover, promoter deletion identified that the upregulated elements lie in -822 to 437 and 263 to -124 and the downregulated elements in -437 to -263 using a beta-galactosidase reporter system. Using electrophoretic mobility shift assay we also found that Rim101p can bind directly with two putative 5'-CCAAGAA-3' sites at the -157 and -629 sites. Interestingly, site-specific mutations show that only the -629 site, not the -157 site, plays an important role in FRP1 expression under iron-limited conditions.

ESCRT-III Protein Snf7 Mediates High-Level Expression of the SUC2 Gene via the Rim101 Pathway

The yeast (Saccharomyces cerevisiae) Snf7 family consists of six highly charged, coiled-coil-forming proteins involved in multivesicular body (MVB) formation. Although all proteins perform a common function at late endosomes, individual mutants also show distinct phenotypes. This suggests that Snf7 homologues have additional functions separate from their role in MVB formation. In this report, we explored the molecular basis for the sucrose-nonfermenting phenotype of snf7 mutants. Our Northern blotting experiments provide evidence that Snf7 is involved in the regulation of SUC2 transcription. The Snf7-dependent regulation of SUC2 transcription does not appear to involve the transcription factor Mig1, since Mig1 phosphorylation after glucose derepression was not affected in a Deltasnf7 mutant. Instead, we show that Snf7 influences SUC2 expression by regulating the level of the transcription factor Nrg1. Snf7 exerts its effects on Nrg1 levels through activation of the transcription factor Rim101, which is part of the yeast alkaline response pathway ("Rim101 pathway"). This is supported by the findings that deletion of RIM101 or overexpression of NRG1 from the ADH1 promoter leads to the same SUC2 expression level as deletion of SNF7. In addition, deletion of other components of the Rim101 pathway, like RIM13 and RIM20, led to the same growth phenotype on raffinose media as deletion of SNF7. Furthermore, Snf7 turned out to be dispensable for SUC2 expression in an NRG1 deletion background. Thus, the effects of Snf7 on SUC2 expression can be completely accounted for by its effect on Nrg1 levels.

Ambient pH signalling in the yeast Yarrowia lipolytica involves YlRim23p/PalC, which interacts with Snf7p/Vps32p, but does not require the long C terminus of YlRim9p/PalI

A conserved ambient pH signal transduction pathway has been evidenced in both ascomycetous yeasts and filamentous fungi, called the Rim or Pal pathway, respectively. However, closely related PalC orthologues are found only in Yarrowia lipolytica and in filamentous fungi, where the Rim9p/PalI factor has a much longer C-terminal tail than in other yeasts. We show here that, like Aspergillus nidulans palI mutants, a Ylrim9Delta mutant has a less extreme phenotype than other mutants of the pathway, whereas rim9 mutants in Saccharomyces cerevisiae and Candida albicans reportedly exhibit a tight Rim phenotype. Deletion of the long C-terminal tail of YlRim9p/PalI had no phenotypic effect on ambient pH signalling. We also show that the Y. lipolytica PalC orthologue, named YlRim23p, is absolutely required for the alkaline pH response. Its only interactant identified in a genome-wide two-hybrid screen is YlSnf7/Vps32p, confirming the link between the Rim and the Vps pathways. YlRim13p and YlRim20p both interact with YlSnf7/Vps32p but not with YlRim23p. The long C-terminal tail of YlRim9p/PalI interacts neither with YlRim23p nor with YlSnf7/Vps32p. These results show that YlRim23p is a bona fide component of the Rim pathway in Y. lipolytica and that it participates in the complexes linking pH signalling and endocytosis.

The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

Biological membranes consist of lipid bilayers. The lipid compositions between the two leaflets of the plasma membrane differ, generating lipid asymmetry. Maintenance of proper lipid asymmetry is physiologically quite important, and its collapse induces several cellular responses including apoptosis and platelet coagulation. Thus, a change in lipid asymmetry must be restored to maintain "lipid asymmetry homeostasis." However, to date no lipid asymmetry-sensing proteins or any related downstream signaling pathways have been identified. We recently demonstrated that expression of the putative yeast sphingoid long-chain base transporter/translocase Rsb1 is induced when glycerophospholipid asymmetry is altered. Using mutant screening, we determined that the pH-responsive Rim101 pathway, the protein kinase Mck1, and the transcription factor Mot3 all act in lipid asymmetry signaling, and that the Rim101 pathway was activated in response to a change in lipid asymmetry. The activated transcription factor Rim101 induces Rsb1 expression via repression of another transcription repressor, Nrg1. Changes in lipid asymmetry are accompanied by cell surface exposure of negatively charged phospholipids; we speculate that the Rim101 pathway recognizes the surface charges.

Adaptation to environmental pH: integrating the Rim101 and calcineurin signal transduction pathways

The ability to appropriately respond to environmental conditions is critical for the survival of simple microbes and for development of complex multicellular organisms. Sensing and responding to a given environmental condition requires the integration of numerous signals through one or more signal transduction pathways. This leads to changes in gene expression, and potentially post-translational modifications, that favour growth in the given environment. In the fungus Candida albicans, an important opportunistic pathogen, environmental pH has profound effects on morphology and proper adaptation to extracellular pH is critical for pathogenesis. Here, we demonstrate that the Rim101/PacC pH-sensing pathway acts in parallel to Crz1, via calcineurin, to adapt to alkaline pH. We also show that the Rim101 pathway acts in parallel to Crz2, independent of calcineurin, to adapt to high lithium concentrations and to repress filamentation at acidic pH. Our studies also revealed a novel requirement for Crz1, Crz2 and calcineurin for growth at acidic pH. From these studies, we propose that the Crz1 homologue Crz2 is calcineurin-independent, but like Crz1, acts in parallel to promote specific Rim101-dependent processes. These results establish and begin to dissect the complex interactions between important signal transduction pathways in C. albicans, which are critical for virulence.

The Transcriptional Response of the Yeast Na+-ATPase ENA1 Gene to Alkaline Stress Involves Three Main Signaling Pathways

Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na(+)-ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5.-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5. end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.

The Ambient pH Response Rim Pathway in Yarrowia lipolytica: Identification of YlRIM9 and Characterization of Its Role in Dimorphism

Yarrowia lipolytica is a dimorphic fungus that secretes either an acidic or an alkaline protease depending on the environmental pH. Previous results have indicated that secretion of the alkaline protease is under control of the pH signaling Pal/Rim pathway originally described in Aspergillus nidulans. Several Y. lipolytica mutants defective in some Rim components of this pathway have been previously isolated and the RIM genes characterized. In the present study, Y. lipolytica RIM9 (palI) gene (YlRIM9) was sequenced from a plasmid (AL414126) of the Genolevures project (the DNA sequence data for YlRIM9 gene has been deposited at EMBL with accession number AJ566902). The derived translation product contains 724 amino acids with a predicted signal peptide and four transmembrane domains in its N-terminal region. We demonstrated that mutation in YlRIM9, as well as in other genes encoding members of the Pal/Rim pathway, did not affect the pH-dependent dimorphic transition of Y. lipolytica. A different pathway must exist in this fungus that controls the effect of pH on dimorphism.

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

The Saccharomyces cerevisiae ynl294cDelta (rim21Delta) 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. rim21Delta 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. rim21Delta and rim101Delta 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. rim21Delta, as well as rim101Delta, was synthetic lethal with slt2Delta, 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, rim21Delta rlm1Delta and rim101Delta rlm1Delta 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.