The transporter PHO84/NtPT1 is a target of aluminum to affect phosphorus absorption in Saccharomyces cerevisiae and Nicotiana tabacum L.

The molecular mechanism of aluminum toxicity in biological systems is not completely understood. Saccharomyces cerevisiae is one of the most used model organisms in the study of environmental metal toxicity. Using an unbiased metallomic approach in yeast, we found that aluminum treatment caused phosphorus deprivation, and the lack of phosphorus increased as the pH of the environment decreased compared to the control strain. By screening the phosphate signaling and response pathway (PHO pathway) in yeast with the synthetic lethality of a new phosphorus-restricted aluminum-sensitive gene, we observed that pho84Δ mutation conferred severe growth defect to aluminum under low-phosphorus conditions, and the addition of phosphate alleviated this sensitivity. Subsequently, the data showed that PHO84 determined the intracellular aluminum-induced phosphorus deficiency, and the expression of PHO84 was positively correlated with aluminum stress, which was mediated by phosphorus through the coordinated regulation of PHO4/PHO2. Moreover, aluminum reduced phosphorus absorption and inhibited tobacco plant growth in acidic media. In addition, the high-affinity phosphate transporter NtPT1 in tobacco exhibited similar effects to PHO84, and overexpression of NtPT1 conferred aluminum resistance in yeast cells. Taken together, positive feedback regulation of the PHO pathway centered on the high-affinity phosphate transporters is a highly conservative mechanism in response to aluminum toxicity. The results may provide a basis for aluminum-resistant microorganisms or plant engineering and acidic soil treatment.

Fungal commensalism modulated by a dual-action phosphate transceptor.

Successful host colonization by fungi in fluctuating niches requires response and adaptation to multiple environmental stresses. However, our understanding about how fungal species thrive in the gastrointestinal (GI) ecosystem by combing multifaceted nutritional stress with respect to homeostatic host-commensal interactions is still in its infancy. Here, we discover that depletion of the phosphate transceptor Pho84 across multiple fungal species encountered a substantial cost in gastrointestinal colonization. Mechanistically, Pho84 enhances the gastrointestinal commensalism via a dual-action activity, coordinating both phosphate uptake and TOR activation by induction of the transcriptional regulator Try4 and downstream commensalism-related transcription. As such, Pho84 promotes Candida albicans commensalism, but this does not translate into enhanced pathogenicity. Thus, our study uncovers a specific nutrient-dependent dual-action regulatory pathway for Pho84 on fungal commensalism.

Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target.

Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor-Sch9-TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor-Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

Temperature preference can bias parental genome retention during hybrid evolution.

Interspecific hybridization can introduce genetic variation that aids in adaptation to new or changing environments. Here, we investigate how hybrid adaptation to temperature and nutrient limitation may alter parental genome representation over time. We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments at 30°C, we identified a number of responses only observed in the colder temperature regime, including the loss of the S. cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid genome. In particular, we discovered a genotype by environment interaction in the form of a loss of heterozygosity event on chromosome XIII; which species' haplotype is lost or maintained is dependent on the parental species' temperature preference and the temperature at which the hybrid was evolved. We show that a large contribution to this directionality is due to a temperature dependent fitness benefit at a single locus, the high affinity phosphate transporter gene PHO84. This work helps shape our understanding of what forces impact genome evolution after hybridization, and how environmental conditions may promote or disfavor the persistence of hybrids over time.

Leishmania amazonensis inorganic phosphate transporter system is increased in the proliferative forms.

Here we characterize a high-affinity Pi transport system energized by a H+ gradient in Leishmania amazonensis. Pi uptake and transcription of LamPho84 gene are differentially regulated during parasite life cycle. Our data suggest that Pi acquisition could be a pivotal task for the success of the parasite throughout its life cycle.

Improvement of selenium enrichment in Rhodotorula glutinis X-20 through combining process optimization and selenium transport.

Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast Rhodotorula glutinis X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from Saccharomyces cerevisiae into R. glutinis X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L-1, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L-1 and 5349.6 µg g-1 (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.

Detecting genetic interactions using parallel evolution in experimental populations.

Eukaryotic genomes contain thousands of genes organized into complex and interconnected genetic interaction networks. Most of our understanding of how genetic variation affects these networks comes from quantitative-trait loci mapping and from the systematic analysis of double-deletion (or knockdown) mutants, primarily in the yeast Saccharomyces cerevisiae. Evolve and re-sequence experiments are an alternative approach for identifying novel functional variants and genetic interactions, particularly between non-loss-of-function mutations. These experiments leverage natural selection to obtain genotypes with functionally important variants and positive genetic interactions. However, no systematic methods for detecting genetic interactions in these data are yet available. Here, we introduce a computational method based on the idea that variants in genes that interact will co-occur in evolved genotypes more often than expected by chance. We apply this method to a previously published yeast experimental evolution dataset. We find that genetic targets of selection are distributed non-uniformly among evolved genotypes, indicating that genetic interactions had a significant effect on evolutionary trajectories. We identify individual gene pairs with a statistically significant genetic interaction score. The strongest interaction is between genes TRK1 and PHO84, genes that have not been reported to interact in previous systematic studies. Our work demonstrates that leveraging parallelism in experimental evolution is useful for identifying genetic interactions that have escaped detection by other methods. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Genetic analysis of suppressor mutants of a pho84 disruptant in the search for genes involved in intracellular inorganic phosphate sensing in Saccharomyces cerevisiae.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

Evolutionary conservation of a core fungal phosphate homeostasis pathway coupled to development in Blastocladiella emersonii.

The model yeast Saccharomyces cerevisiae elicits a transcriptional response to phosphate (Pi) depletion. To determine the origins of the phosphate response (PHO) system, we bioinformatically identified putative PHO components in the predicted proteomes of diverse fungi. Our results suggest that the PHO system is ancient; however, components have been expanded or lost in different fungal lineages. To show that a similar physiological response is present in deeply-diverging fungi we examined the transcriptional and physiological response of PHO genes to Pi depletion in the blastocladiomycete Blastocladiella emersonii. Our physiological experiments indicate that B. emersonii relies solely on high-affinity Na+-independent Pho84-like transporters. In response to Pi depletion, BePho84 paralogues were 4-8-fold transcriptionally upregulated, whereas several other PHO homologues like phosphatases and vacuolar transporter chaperone (VTC) complex components show 2-3-fold transcriptional upregulation. Since Pi has been shown to be important during the development of B. emersonii, we sought to determine if PHO genes are differentially regulated at different lifecycle stages. We demonstrate that a similar set of PHO transporters and phosphatases are upregulated at key points during B. emersonii development. Surprisingly, some genes upregulated during Pi depletion, including VTC components, are repressed at these key stages of development indicating that PHO genes are regulated by different pathways in different developmental and environmental situations. Overall, our findings indicate that a complex PHO network existed in the ancient branches of the fungi, persists in diverse extant fungi, and that this ancient network is likely to be involved in development and cell cycle regulation.

Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling.

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

Dual role of starvation signaling in promoting growth and recovery.

Growing cells are subject to cycles of nutrient depletion and repletion. A shortage of nutrients activates a starvation program that promotes growth in limiting conditions. To examine whether nutrient-deprived cells prepare also for their subsequent recovery, we followed the transcription program activated in budding yeast transferred to low-phosphate media and defined its contribution to cell growth during phosphate limitation and upon recovery. An initial transcription wave was induced by moderate phosphate depletion that did not affect cell growth. A second transcription wave followed when phosphate became growth limiting. The starvation program contributed to growth only in the second, growth-limiting phase. Notably, the early response, activated at moderate depletion, promoted recovery from starvation by increasing phosphate influx upon transfer to rich medium. Our results suggest that cells subject to nutrient depletion prepare not only for growth in the limiting conditions but also for their predicted recovery once nutrients are replenished.

The nutrient transceptor/PKA pathway functions independently of TOR and responds to leucine and Gcn2 in a TOR-independent manner.

Two nutrient-controlled signalling pathways, the PKA and TOR pathway, play a major role in nutrient regulation of growth as well as growth-correlated properties in yeast. The relationship between the two pathways is not well understood. We have used Gap1 and Pho84 transceptor-mediated activation of trehalase and phosphorylation of fragmented Sch9 as a read-out for rapid nutrient activation of PKA or TORC1, respectively. We have identified conditions in which L-citrulline-induced activation of Sch9 phosphorylation is compromised, but not activation of trehalase: addition of the TORC1 inhibitor, rapamycin and low levels of L-citrulline. The same disconnection was observed for phosphate activation in phosphate-starved cells. The leu2 auxotrophic mutation reduces amino acid activation of trehalase, which is counteracted by deletion of GCN2. Both effects were also independent of TORC1. Our results show that rapid activation of the TOR pathway by amino acids is not involved in rapid activation of the PKA pathway and that effects of Gcn2 inactivation as well as leu2 auxotrophy all act independently of the TOR pathway. Hence, rapid nutrient signalling to PKA and TOR in cells arrested by nutrient starvation acts through parallel pathways.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.

The biochemical characterization of two phosphate transport systems in Phytomonas serpens.

Inorganic phosphate (Pi) is an essential nutrient for all organisms because it is required for a variety of biochemical processes, such as signal transduction and the synthesis of phosphate-containing biomolecules. Assays of 32Pi uptake performed in the absence or in the presence of Na+ indicated the existence of a Na+-dependent and a Na+-independent Pi transporter in Phytomonas serpens. Phylogenetic analysis of two hypothetical protein sequences of Phytomonas (EM1) showed similarities to the high-affinity Pi transporters of Saccharomyces cerevisiae: Pho84, a Na+-independent Pi transporter, and Pho89, a Na+-dependent Pi transporter. Plasma membrane depolarization by FCCP, an H+ ionophore, strongly decreased Pi uptake via both Na+-independent and Na+-dependent carriers, indicating that a membrane potential is essential for Pi influx. In addition, the furosemide-sensitive Na+-pump activity in the cells grown in low Pi conditions was found to be higher than the activity detected in the plasma membrane of cells cultivated at high Pi concentration, suggesting that the up-regulation of the Na+-ATPase pump could be related to the increase of Pi uptake by the Pho89p Na+:Pi symporter. Here we characterize for the first time two inorganic phosphate transporters powered by Na+ and H+ gradients and activated by low Pi availability in the phytopathogen P. serpens.

The conservation of phosphate-binding residues among PHT1 transporters suggests that distinct transport affinities are unlikely to result from differences in the phosphate-binding site.

The plant PHosphate Transporter 1 (PHT1) family of membrane proteins belongs to the major facilitator super family and plays a major role in the acquisition of inorganic phosphate (Pi) from the soil and its transport within the plant. These transporters have been well characterized for expression patterns, localization, and in some cases affinity. Furthermore, the crystal structure of a high-affinity eukaryotic phosphate transporter from the fungus Piriformospora indica (PiPT) has revealed important information on the residues involved in Pi transport. Using multiple-sequence alignments and homology modelling, the phosphate-binding site residues were shown to be well conserved between all the plant PHT1 proteins, Saccharomyces cerevisiae PHO84 and PiPT. For example, Asp 324 in PiPT is conserved in the equivalent position in all plant PHT1 and yeast transporters analyzed, and this residue in ScPHO84 was shown by mutagenesis to be important for both the binding and transport of Pi. Moreover, Asp 45 and Asp 149, which are predicted to be involved in proton import, and Lys 459, which is putatively involved in Pi-binding, are all fully conserved in PHT1 and ScPHO84 transporters. The conserved nature of the residues that play a key role in Pi-binding and transport across the PHT1 family suggests that the differing Pi affinities of these transporters do not reside in differences in the Pi-binding site. Recent studies suggest that phosphate transporters could possess dual affinity and that post-translational modifications may be important in regulating affinity for phosphate.

Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

Inorganic Phosphate and Sulfate Transport in S. cerevisiae.

Inorganic ions such as phosphate and sulfate are essential macronutrients required for a broad spectrum of cellular functions and their regulation. In a constantly fluctuating environment microorganisms have for their survival developed specific nutrient sensing and transport systems ensuring that the cellular nutrient needs are met. This chapter focuses on the S. cerevisiae plasma membrane localized transporters, of which some are strongly induced under conditions of nutrient scarcity and facilitate the active uptake of inorganic phosphate and sulfate. Recent advances in studying the properties of the high-affinity phosphate and sulfate transporters by means of site-directed mutagenesis have provided further insight into the molecular mechanisms contributing to substrate selectivity and transporter functionality of this important class of membrane transporters.

The Phosphate Transporter Gene OsPht1;4 Is Involved in Phosphate Homeostasis in Rice.

A total of 13 phosphate transporters in rice (Oryza sative) have been identified as belonging to the Pht1 family, which mediates inorganic phosphate (Pi) uptake and transport. We report the biological property and physiological role of OsPht1;4 (OsPT4). Overexpressing OsPT4 resulted in significant higher Pi accumulation in roots, straw and brown rice, and suppression of OsPT4 caused decreased Pi concentration in straw and brown rice. Expression of the ß-glucuronidase reporter gene driven by the OsPT4 promoter showed that OsPT4 is expressed in roots, leaves, ligules, stamens, and caryopses under sufficient Pi conditions, consistent with the expression profile showing that OsPT4 has high expression in roots and flag leaves. The transcript level of OsPT4 increased significantly both in shoots and roots with a long time Pi starvation. OsPT4 encoded a plasma membrane-localized protein and was able to complement the function of the Pi transporter gene PHO84 in yeast. We concluded that OsPT4 is a functional Pi-influx transporter involved in Pi absorption in rice that might play a role in Pi translocation. This study will enrich our understanding about the physiological function of rice Pht1 family genes.

Coregulated expression of the Na+/phosphate Pho89 transporter and Ena1 Na+-ATPase allows their functional coupling under high-pH stress.

The yeast Saccharomyces cerevisiae has two main high-affinity inorganic phosphate (Pi) transporters, Pho84 and Pho89, that are functionally relevant at acidic/neutral pH and alkaline pH, respectively. Upon Pi starvation, PHO84 and PHO89 are induced by the activation of the PHO regulon by the binding of the Pho4 transcription factor to specific promoter sequences. We show that PHO89 and PHO84 are induced by alkalinization of the medium with different kinetics and that the network controlling Pho89 expression in response to alkaline pH differs from that of other members of the PHO regulon. In addition to Pho4, the PHO89 promoter is regulated by the transcriptional activator Crz1 through the calcium-activated phosphatase calcineurin, and it is under the control of several repressors (Mig2, Nrg1, and Nrg2) coordinately regulated by the Snf1 protein kinase and the Rim101 transcription factor. This network mimics the one regulating expression of the Na(+)-ATPase gene ENA1, encoding a major determinant for Na(+) detoxification. Our data highlight a scenario in which the activities of Pho89 and Ena1 are functionally coordinated to sustain growth in an alkaline environment.