Transcription Factor Pho4 Research Articles

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5.

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4's release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

The nutrient-responsive CDK Pho85 primes the Sch9 kinase for its activation by TORC1.

Yeast cells maintain an intricate network of nutrient signaling pathways enabling them to integrate information on the availability of different nutrients and adjust their metabolism and growth accordingly. Cells that are no longer capable of integrating this information, or that are unable to make the necessary adaptations, will cease growth and eventually die. Here, we studied the molecular basis underlying the synthetic lethality caused by loss of the protein kinase Sch9, a key player in amino acid signaling and proximal effector of the conserved growth-regulatory TORC1 complex, when combined with either loss of the cyclin-dependent kinase (CDK) Pho85 or loss of its inhibitor Pho81, which both have pivotal roles in phosphate sensing and cell cycle regulation. We demonstrate that it is specifically the CDK-cyclin pair Pho85-Pho80 or the partially redundant CDK-cyclin pairs Pho85-Pcl6/Pcl7 that become essential for growth when Sch9 is absent. Interestingly, the respective three CDK-cyclin pairs regulate the activity and distribution of the phosphatidylinositol-3 phosphate 5-kinase Fab1 on endosomes and vacuoles, where it generates phosphatidylinositol-3,5 bisphosphate that serves to recruit both TORC1 and its substrate Sch9. In addition, Pho85-Pho80 directly phosphorylates Sch9 at Ser726, and to a lesser extent at Thr723, thereby priming Sch9 for its subsequent phosphorylation and activation by TORC1. The TORC1-Sch9 signaling branch therefore integrates Pho85-mediated information at different levels. In this context, we also discovered that loss of the transcription factor Pho4 rescued the synthetic lethality caused by loss of Pho85 and Sch9, indicating that both signaling pathways also converge on Pho4, which appears to be wired to a feedback loop involving the high-affinity phosphate transporter Pho84 that fine-tunes Sch9-mediated responses.

Reb1, Cbf1, and Pho4 Bias Histone Sliding and Deposition Away from Their Binding Sites.

In transcriptionally active genes, nucleosome positions in promoters are regulated by nucleosome-displacing factors (NDFs) and chromatin-remodeling enzymes. Depletion of NDFs or the RSC chromatin remodeler shrinks or abolishes the nucleosome-depleted regions (NDRs) in promoters, which can suppress gene activation and result in cryptic transcription. Despite their vital cellular functions, how the action of chromatin remodelers may be directly affected by site-specific binding factors like NDFs is poorly understood. Here, we demonstrate that two NDFs, Reb1 and Cbf1, can direct both Chd1 and RSC chromatin-remodeling enzymes in vitro, stimulating repositioning of the histone core away from their binding sites. Interestingly, although the Pho4 transcription factor had a much weaker effect on nucleosome positioning, both NDFs and Pho4 were able to similarly redirect positioning of hexasomes. In chaperone-mediated nucleosome assembly assays, Reb1 but not Pho4 showed an ability to block deposition of the histone H3/H4 tetramer, but Reb1 did not block addition of the H2A/H2B dimer to hexasomes. Our in vitro results show that NDFs bias the action of remodelers to increase the length of the free DNA in the vicinity of their binding sites. These results suggest that NDFs could directly affect NDR architecture through chromatin remodelers.

Short tandem repeats recruit transcription factors to tune eukaryotic gene expression

Transcription factors (TFs) regulate gene expression via sequence-specific binding to regulatory elements in genomic DNA. Short tandem repeats (STRs) are enriched in these regulatory elements and have been widely associated with changes in gene expression, but the mechanism by which STRs modulate expression remains unknown. To address this gap, we leveraged the MITOMI microfluidic platform to measure equilibrium binding and kinetics of basic-helix-loop-helix TFs Pho4 and MAX to their consensus binding sites, surrounded by various random or repetitive sequences.

Phosphate Starvation by Energy Metabolism Disturbance in Candida albicansvip1Δ/Δ Induces Lipid Droplet Accumulation and Cell Membrane Damage.

Phosphorus in the form of phosphate (Pi) is an essential element for metabolic processes, including lipid metabolism. In yeast, the inositol polyphosphate kinase vip1 mediated synthesis of inositol heptakisphosphate (IP7) regulates the phosphate-responsive (PHO) signaling pathway, which plays an important role in response to Pi stress. The role of vip1 in Pi stress and lipid metabolism of Candida albicans has not yet been studied. We found that when vip1Δ/Δ was grown in glucose medium, if Pi was supplemented in the medium or mitochondrial Pi transporter was overexpressed in the strain, the lipid droplet (LD) content was reduced and membrane damage was alleviated. However, further studies showed that neither the addition of Pi nor the overexpression of the Pi transporter affected the energy balance of vip1Δ/Δ. In addition, the LD content of vip1Δ/Δ grown in Pi limitation medium PNMC was lower than that grown in SC, and the metabolic activity of vip1Δ/Δ grown in PNMC was also lower than that grown in SC medium. This suggests that the increase in Pi demand by a high energy metabolic rate is the cause of LD accumulation in vip1Δ/Δ. In addition, in the vip1Δ/Δ strains, the core transcription factor PHO4 in the PHO pathway was transported to the vacuole and degraded, which reduced the pathway activity. However, this does not mean that knocking out vip1 completely blocks the activation of the PHO pathway, because the LD content of vip1Δ/Δ grown in the medium with β-glycerol phosphate as the Pi source was significantly reduced. In summary, the increased Pi demand and the decreased PHO pathway activity in vip1Δ/Δ ultimately lead to LD accumulation and cell membrane damage.

Functional analysis of a Phytophthora host-translocated effector using the yeast model system.

Background Phytophthora plant pathogens secrete effector proteins that are translocated into host plant cells during infection and collectively contribute to pathogenicity. A subset of these host-translocated effectors can be identified by the amino acid motif RXLR (arginine, any amino acid, leucine, arginine). Bioinformatics analysis has identified hundreds of putative RXLR effector genes in Phytophthora genomes, but the specific molecular function of most remains unknown.MethodsHere we describe initial studies to investigate the use of Saccharomyces cerevisiae as a eukaryotic model to explore the function of Phytophthora RXLR effector proteins.Results and ConclusionsExpression of individual RXLR effectors in yeast inhibited growth, consistent with perturbation of a highly conserved cellular process. Transcriptome analysis of yeast cells expressing the poorly characterized P. sojae RXLR effector Avh110 identified nearly a dozen yeast genes whose expression levels were altered greater than two-fold compared to control cells. All five of the most down-regulated yeast genes are normally induced under low phosphate conditions via the PHO4 transcription factor, indicating that PsAvh110 perturbs the yeast regulatory network essential for phosphate homeostasis and suggesting likely PsAvh110 targets during P. sojae infection of its soybean host.

Lack of 14-3-3 proteins in Saccharomyces cerevisiae results in cell-to-cell heterogeneity in the expression of Pho4-regulated genes SPL2 and PHO84

BackgroundIon homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes involved in high-affinity phosphate uptake (PHO genes) are strongly induced during both phosphate and potassium starvation, indicating a link between phosphate and potassium homeostasis. However, the signal transduction processes involved are not completely understood. As 14-3-3 proteins are key regulators of signal transduction processes, we investigated the effect of deletion of the 14-3-3 genes BMH1 or BMH2 on gene expression during potassium starvation and focused especially on the expression of genes involved in phosphate uptake.ResultsGenome-wide analysis of the effect of disruption of either BMH1 or BMH2 revealed that the mRNA levels of the PHO genes PHO84 and SPL2 are greatly reduced in the mutant strains compared to the levels in wild type strains. This was especially apparent at standard potassium and phosphate concentrations. Furthermore the promoter of these genes is less active after deletion of BMH1. Microscopic and flow cytometric analysis of cells with GFP-tagged SPL2 showed that disruption of BMH1 resulted in two populations of genetically identical cells, cells expressing the protein and the majority of cells with no detectible expression. Heterogeneity was also observed for the expression of GFP under control of the PHO84 promoter. Upon deletion of PHO80 encoding a regulator of the transcription factor Pho4, the effect of the BMH1 deletion on SPL2 and PHO84 promoter was lost, suggesting that the BMH1 deletion mainly influences processes upstream of the Pho4 transcription factor.ConclusionOur data indicate that that yeast cells can be in either of two states, expressing or not expressing genes required for high-affinity phosphate uptake and that 14-3-3 proteins are involved in the process(es) that establish the activation state of the PHO regulon.

Arsenic inorganic compounds cause oxidative stress mediated by the transcription factor PHO4 in Candida albicans

Arsenic is a toxic metalloid widespread in nature. Recently, it has been demonstrated a main role of the transcription factor Pho4 in the acquisition of tolerance to arsenic-derived compounds, arsenite and arsenate in Candida albicans. Here, the effect of these compounds on this pathogenic yeast has been analyzed. In wild type cells, both arsenite and arsenate induced a marked increase in the endogenous production of Reactive Oxygen Species (ROS), together with the accumulation of intracellular trehalose and the activation of catalase, suggesting their role as generators of oxidative stress in this yeast. However, a pho4 null mutant showed a minor increase of intracellular ROS and a different kinetics of catalase activation upon exposure to arsenite and arsenate. Interestingly, the enzymatic activity of glutathione reductase and superoxide dismutase were exclusively triggered by arsenite but not by arsenate. pho4 mutant cells were also found to be sensitive to azide but significantly resistant to arsenate through a process dependent on an active electron transport chain and the alternative oxidase system. Therefore, arsenic-derived compounds induce a strong antioxidant response in C.albicans via different mechanisms.

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient Pi. Pi-limitation induces upregulation of inositol heptakisphosphate (IP7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Furthermore, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of Pi starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.

Evolution of reduced co-activator dependence led to target expansion of a starvation response pathway.

Although combinatorial regulation is a common feature in gene regulatory networks, how it evolves and affects network structure and function is not well understood. In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are required for gene induction and survival during phosphate starvation. In the related human commensal C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate starvation and is only partially required for inducing PHO genes. Phylogenetic survey suggests that reduced dependence on Pho2 evolved in C. glabrata and closely related species. In S. cerevisiae, less Pho2-dependent Pho4 orthologs induce more genes. In C. glabrata, its Pho4 binds to more locations and induces three times as many genes as Pho4 in S. cerevisiae does. Our work shows how evolution of combinatorial regulation allows for rapid expansion of a gene regulatory network's targets, possibly extending its physiological functions.

Pho4 Is Essential for Dissemination of Cryptococcus neoformans to the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH.

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. Cryptococcus neoformans disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain (hlh3Δ/pho4Δ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. The pho4Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, the pho4Δ mutant had a growth defect and compromised phosphate uptake. The pho4Δ mutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. The pho4Δ mutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent. IMPORTANCE Cryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen, C.neoformans must obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant (pho4Δ mutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of the pho4Δ mutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood.

The Candida albicans Pho4 Transcription Factor Mediates Susceptibility to Stress and Influences Fitness in a Mouse Commensalism Model.

The Pho4 transcription factor is required for growth under low environmental phosphate concentrations in Saccharomyces cerevisiae. A characterization of Candida albicans pho4 mutants revealed that these cells are more susceptible to both osmotic and oxidative stress and that this effect is diminished in the presence of 5% CO2 or anaerobiosis, reflecting the relevance of oxygen metabolism in the Pho4-mediated response. A pho4 mutant was as virulent as wild type strain when assayed in the Galleria mellonella infection model and was even more resistant to murine macrophages in ex vivo killing assays. The lack of Pho4 neither impairs the ability to colonize the murine gut nor alters the localization in the gastrointestinal tract. However, we found that Pho4 influenced the colonization of C. albicans in the mouse gut in competition assays; pho4 mutants were unable to attain high colonization levels when inoculated simultaneously with an isogenic wild type strain. Moreover, pho4 mutants displayed a reduced adherence to the intestinal mucosa in a competitive ex vivo assays with wild type cells. In vitro competitive assays also revealed defects in fitness for this mutant compared to the wild type strain. Thus, Pho4, a transcription factor involved in phosphate metabolism, is required for adaptation to stress and fitness in C. albicans.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

The transcription factor GCN4 regulates PHM8 and alters triacylglycerol metabolism in Saccharomyces cerevisiae.

PHM8 is a very important enzyme in nonpolar lipid metabolism because of its role in triacylglycerol (TAG) biosynthesis under phosphate stress conditions. It is positively regulated by the PHO4 transcription factor under low phosphate conditions; however, its regulation has not been explored under normal physiological conditions. General control nonderepressible (GCN4), a basic leucine-zipper transcription factor activates the transcription of amino acids, purine biosynthesis genes and many stress response genes under various stress conditions. In this study, we demonstrate that the level of TAG is regulated by the transcription factor GCN4. GCN4 directly binds to its consensus recognition sequence (TGACTC) in the PHM8 promoter and controls its expression. The analysis of cells expressing the P PHM8 -lacZ reporter gene showed that mutations (TGACTC-GGGCCC) in the GCN4-binding sequence caused a significant increase in β-galactosidase activity. Mutation in the GCN4 binding sequence causes an increase in PHM8 expression, lysophosphatidic acid phosphatase activity and TAG level. PHM8, in conjunction with DGA1, a mono- and diacylglycerol transferase, controls the level of TAG. These results revealed that GCN4 negatively regulates PHM8 and that deletion of GCN4 causes de-repression of PHM8, which is responsible for the increased TAG content in gcn4∆ cells.

PHO4 transcription factor regulates triacylglycerol metabolism under low-phosphate conditions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, PHM8 encodes a phosphatase that catalyses the dephosphorylation of lysophosphatidic acids to monoacylglycerol and nucleotide monophosphate to nucleoside and releases free phosphate. In this report, we investigated the role of PHM8 in triacylglycerol metabolism and its transcriptional regulation by a phosphate responsive transcription factor Pho4p under low-phosphate conditions. We found that the wild-type (BY4741) cells accumulate triacylglycerol and the expression of PHM8 was high under low-phosphate conditions. Overexpression of PHM8 in the wild-type, phm8Δ and quadruple phosphatase mutant (pah1Δdpp1Δlpp1Δapp1Δ) caused an increase in the triacylglycerol levels. However, the introduction of the PHM8 deletion into the quadruple phosphatase mutant resulted in a reduction in triacylglycerol levels and LPA phosphatase activity. The transcriptional activator Pho4p binds to the PHM8 promoter under low-phosphate conditions, activating PHM8 expression, which leads to the formation of monoacylglycerol from LPA. The synthesized monoacylglycerol is acylated to diacylglycerol by Dga1p, which is further acylated to triacylglycerol by the same enzyme.

The PHO4 Transcription Factor Regulates Triacylglycerol Metabolism under Low Phosphate Conditions in Saccharomyces cerevisiae

ObjectiveTo study the transcriptional regulation of lipid metabolism in Saccharomyces cerevisiae under low phosphate (Pi) conditions.Methodology[14C]Acetate labeling, confocal microscopy, gas chromatography, semi quantitative and quantitative PCR. Electromobility shift assay (EMSA) and β‐galactosidase assay. To validate the binding site‐directed mutagenesis was performed. Enzyme kinetics and characterization.ResultsThe expression of PHM8 is high under low phosphate conditions, which encodes a LPA phosphatase and nucleotidase. In an electrophoretic mobility shift assay, Pho4p was shown to bind specifically in PHM8 promoter. Analyses of PHM8 mRNA, LPA phosphatase activity and triacylglycerol levels under low phosphate conditions in phm8Δ showed that the plasmid harbouring mutated promoter along with the PHM8 gene had much lower PHM8 expression than the plasmid harbouring the native promoter with the gene. Enzyme kinetics with the differentially phosphorylated Phm8p using LPA and CMP as substrates showed that LPA is the preferred over CMP under low phosphate conditions. The results indicate that the transcriptional activator Pho4p binds to the PHM8 promoter under low phosphate conditions, activating PHM8 expression and leading to the formation of monoacylglycerol, which is successively acylated by Dga1p to form diacylglycerol and triacylglycerol. imageConclusionThe phosphate‐mediated transcriptional activation of PHM8 by Pho4p affects triacylglycerol biosynthesis by controlling lysophosphatidic acid and MAG flux.

The Pho4 transcription factor mediates the response to arsenate and arsenite in Candida albicans.

Arsenate (As (V)) is the dominant form of the toxic metalloid arsenic (As). Microorganisms have consequently developed mechanisms to detoxify and tolerate this kind of compounds. In the present work, we have explored the arsenate sensing and signaling mechanisms in the pathogenic fungus Candida albicans. Although mutants impaired in the Hog1 or Mkc1-mediated pathways did not show significant sensitivity to this compound, both Hog1 and Mkc1 became phosphorylated upon addition of sodium arsenate to growing cells. Hog1 phosphorylation upon arsenate challenge was shown to be Ssk1-dependent. A screening designed for the identification of transcription factors involved in the arsenate response identified Pho4, a transcription factor of the myc-family, as pho4 mutants were susceptible to As (V). The expression of PHO4 was shortly induced in the presence of sodium arsenate in a Hog1-independent manner. Pho4 level affects Hog1 phosphorylation upon As (V) challenge, suggesting an indirect relationship between Pho4 activity and signaling in C. albicans. Pho4 also mediates the response to arsenite as revealed by the fact that pho4 defective mutants are sensitive to arsenite and Pho4 becomes phosphorylated upon sodium arsenite addition. Arsenite also triggers Hog1 phosphorylation by a process that is, in this case, independent of the Ssk1 kinase. These results indicate that the HOG pathway mediates the response to arsenate and arsenite in C. albicans and that the Pho4 transcription factor can differentiate among As (III), As (V) and Pi, triggering presumably specific responses.

The regulators of yeast PHO system participate in the transcriptional regulation of G1 cyclin under alkaline stress conditions.

The yeast Pho85 kinase oversees whether environmental conditions are favourable for cell growth and enables yeast cells to express only genes that are appropriate for the conditions. Alkaline stress perturbs transport of molecules across the plasma membrane that is vital for cell survival. Progression through the cell cycle is halted until the cells can adapt to the stress conditions. I found that Pho85 is required for CLN2 expression and that overproduction of the transcription factors Pho4, Rim101 and Crz1, all targets of Pho85, inhibited CLN2 expression. CLN2 expression in the absence of Pho85 could be recovered only when all the three transcription factors were deleted. Whi5, a functional homologue of the mammalian Rb protein, represses CLN2 expression and is inactivated when phosphorylated by either of the CDK-cyclin complexes, Cdc28-Cln3 or Pho85-Pcl9. Under alkaline conditions, the absence of Whi5 caused an increase in CLN2 expression but failed to do so when Pho85 was also absent, or when Pho4 was overproduced. The expression level of CLN2 in a Δpho85 Δpho4 Δrim101 Δcrz1 quadruple mutant was stimulated when the Whi5 activity was repressed by overproduction of Pho85-Pcl9. These results indicate that Whi5 is also under control of alkaline stress. The inhibitory function of Whi5 on CLN2 is dependent on Rpd3 HDAC, and the absence of Rpd3 could also suppress the inhibitory effect of Pho4 overproduction. Based on these findings, a model is presented in which Pho85 and Pho4 functions in CLN2 regulation under alkaline conditions.