Biochemical Journal

Research article

Differential roles for the low-affinity phosphate transporters Pho87 and Pho90 in Saccharomyces cerevisiae

Ruben Ghillebert, Erwin Swinnen, Pepijn De Snijder, Bart Smets, Joris Winderickx

Abstract

When starved of Pi, yeast cells activate the PHO signalling pathway, wherein the Pho4 transcription factor mediates expression of genes involved in Pi acquisition, such as PHO84, encoding the high-affinity H+/Pi symporter. In contrast, transcription of PHO87 and PHO90, encoding the low-affinity H+/Pi transport system, is independent of phosphate status. In the present work, we reveal that, upon Pi starvation, these low-affinity Pi transporters are endocytosed and targeted to the vacuole. For Pho87, this process strictly depends on SPL2, another Pho4-dependent gene that encodes a protein known to interact with the N-terminal SPX domain of the transporter. In contrast, the vacuolar targeting of Pho90 upon Pi starvation is independent of both Pho4 and Spl2, although it still requires its SPX domain. Furthermore, both Pho87 and Pho90 are also targeted to the vacuole upon carbon-source starvation or upon treatment with rapamycin, which mimics nitrogen starvation, but although these responses are independent of PHO pathway signalling, they again require the N-terminal SPX domain of the transporters. These observations suggest that other SPX-interacting proteins must be involved. In addition, we show that Pho90 is the most important Pi transporter under high Pi conditions in the absence of a high-affinity Pi-transport system. Taken together, our results illustrate that Pho87 and Pho90 represent non-redundant Pi transporters, which are tuned by the integration of multiple nutrient signalling mechanisms in order to adjust Pi-transport capacity to the general nutritional status of the environment.

  • nutrient signalling
  • phosphate transporter
  • Pho87
  • Pho90
  • Scaccharomyces cerevisiae
  • vacuolar targeting

INTRODUCTION

As Pi is an essential nutrient for all organisms, maintaining proper Pi homoeostasis is critical to ensure survival of cells and organisms. A crucial step in controlling Pi homoeostasis encompasses the transport of Pi across the plasma membrane into the cell. Since cellular membranes are impermeable to Pi, specialized integral membrane proteins are required to transport this essential compound. In Saccharomyces cerevisiae, two Pi-symport systems exist [13]. The low-affinity transport system has a Km for external Pi of approx. 1 mM and satisfies the cellular need for Pi at normal or high Pi concentrations. This low-affinity Pi-transport system is believed to be composed of the partially redundant Pho87 and Pho90 H+/Pi symporters [4]. A third gene, PHO91, was initially considered to be a third component of the low-affinity plasma membrane Pi-transport system [5], but later studies localized the Pho91 protein at the vacuolar membrane, indicating that this protein is not directly involved in the acquisition of Pi from the external medium [6]. The high-affinity Pi-transport system has a Km of approx. 10 μM and is sustained by the Pho84 and Pho89 high-affinity H+/Pi and Na+/Pi symporters [7,8]. Although expression of the PHO87, PHO90 and PHO91 genes was shown to be independent of Pi concentrations, transcription of PHO84 and PHO89 is induced when Pi becomes limiting, a process controlled by the PHO pathway [9]. Signal flow through this PHO pathway is regulated by the CKI (cyclin-dependent kinase inhibitor) Pho81. Under Pi-starving conditions, activation of this CKI leads to inhibition of the Pho85–Pho80 cyclin-dependent kinase complex and nuclear translocation of the dephosphorylated transcription factor Pho4. Nuclear Pho4 then induces a set of genes known as the PHO genes [10], which includes PHO84 and PHO89. When sufficient amounts of extracellular Pi are available, Pho81 is inactivated and this results in an active Pho85–Pho80 kinase complex that is able to phosphorylate and trigger the nuclear exclusion of Pho4, with the consequent repression of the PHO genes [11].

An additional role for the Pho85–Pho80 complex is to regulate the localization and hence the activation of the Rim15 protein kinase in response to fluctuating Pi levels [11]. Rim15 is essential for proper entry into stationary phase as it controls the activity of the transcription factors Msn2, Msn4 and Gis1, which are required for expression of the so-called STRE (stress-responsive element) genes and the PDS (post-diauxic shift) genes respectively [1214]. Rim15 acts as a nutritional integrator in yeast, since it is regulated by at least four nutrient-regulated kinases: PKA (protein kinase A) inhibits Rim15 activity [15], whereas TORC1 [TOR (target of rapamycin) complex 1] and Sch9 promote cytoplasmic retention [16,17] and the Pho85–Pho80 complex promotes its nuclear exclusion [11,18]. Under Pi-starving conditions, inhibition of the Pho85–Pho80 kinase by Pho81 results in an active nuclear Rim15 and proper entry into stationary phase. In contrast, under high Pi conditions, phosphorylation of Rim15 by the Pho85–Pho80 kinase induces its nuclear exclusion thereby leading to its inactivation. In line with this, we have already shown that the Pho85–Pho80 complex mediates Pi-dependent activation of typical PKA targets, such as activation of trehalase, mobilization of trehalose and repression of stress-responsive genes, and that the regulation of many of these properties occurs via a Pho85–Pho80-dependent inhibition of Rim15 activity [19].

The ability of Pi to act as a nutrient signal affecting PKA targets is closely correlated with its uptake into the cell, as inactivation of Pi transport by destruction of the proton gradient across the plasma membrane with the protonophore 2,4-dinitrophenol completely prevented Pi-induced trehalase activation [20]. Although the predominant Pi-transport activity present in Pi-starved cells is represented by the Pho84 transporter [7,21], which is additionally exemplified by the inability of a pho84Δ strain to mediate rapid Pi signalling [22,23], we previously observed clear activation of trehalase after addition of Pi to cells deleted for PHO4 or PHO81, in which no PHO84 transcription can be detected [19]. Additionally, both Pho84 and Pho87 have been shown to act as specific Pi sensors for rapid Pi signalling [20], but the genetic and physiological relevance of Pi signalling through Pho87 has never been investigated. Our results reveal that, in the absence of Pho4, the low-affinity Pi transporter Pho87 becomes essential for Pi signalling. We show that in Pi-starved cells, the low-affinity Pi transporters Pho87 and Pho90 are normally subjected to vacuolar targeting, but in cells devoid of Pho4 activity, this process is specifically hampered for Pho87 due to the lack of expression of SPL2, which was previously identified as a negative regulator of low-affinity Pi transport and was shown to interact with the N-terminal SPX domain of both Pho87 and Pho90 [4,24]. However, we now demonstrate that, in contrast with Pho87, the vacuolar targeting of the Pho90 upon Pi starvation is independent of both Pho4 activity and Spl2. Furthermore, when cells are starved of other essential nutrients or are treated with rapamycin, the vacuolar targeting applies to both low-affinity Pi transporters, and this process is also independent of Pho4 and Spl2. Nonetheless, the vacuolar targeting of both Pho87 and Pho90 under these conditions still depends on their N-terminal SPX domain. This indicates that, besides Spl2, other, yet unidentified, proteins must interact with this domain in order to fine-tune the presence of the low-affinity Pi transporters at the plasma membrane in response to a multitude of nutrient signals.

EXPERIMENTAL

Strains

S. cerevisiae strains used in the present study are listed in Table 1. Deletions were made using PCR-based disruption cassettes as described previously [25]. Yeast was transformed with the Gietz method [26]. Sporulation was performed by spotting diploid cells on to plates containing 1% potassium acetate and 0.1% KHCO3, pH 6.0, for 5–6 days at 24 °C. Tetrads were dissected and separated in a raster using a micromanipulator (Singer Instruments). The strains PTEF-EGFP-PHO87, PTEF-EGFP-PHO90, PTEF-EGFP-PHO87Δ375N and PTEF-EGFP-PHO90Δ375N (where EGFP is enhanced green fluoresecent protein) were kindly provided by Hürlimann et al. [4,6].

View this table:
Table 1 Strains used in the present study

WT, wild-type; Y.K.O., yeast knockout library.

Culture and starvation conditions

Yeast cells were grown in ScD [Sc (synthetic complete) minimal medium containing 0.5% (NH4)2SO4 and 1.9 g/l yeast nitrogen base (without amino acids), supplemented with all amino acids/nucleotides (Complete Synthetic Medium, MP Biomedicals) and 2% (w/v) glucose]. For monitoring growth on ScD agar with fluctuating extracellular Pi levels, a preculture on ScD was inoculated into fresh ScD medium at an initial D600 of 0.1 and 10-fold dilutions were spotted on to Pi-starvation medium (5.7 g/l of yeast nitrogen base with 0.5% ammonium sulfate and without Pi) with 2% (w/v) glucose supplemented with all amino acids/nucleotides (Complete Synthetic Medium, MP Biomedicals) and with a known Pi concentration. Pictures were taken after 2–3 days. For growth experiments with arsenate, cells were grown in YPD [standard rich medium with 2% (w/v) glucose] and spotted on to YPD agar or on to YPD agar with an additional 10 mM Pi and with or without 5 mM AsO4. For Pi signalling and consumption experiments, yeast cells were grown into mid-exponential phase (D600 of 1.5–2) on ScD medium, harvested, washed twice and transferred to Pi-starvation medium with 4% (w/v) glucose and required amino acid/nucleotide supplements (Complete Synthetic Medium, MP Biomedicals). Cells were Pi-starved for 3 days, during which the starvation medium was refreshed daily. Before re-addition of Pi, the Pi-starved glucose-repressed cells were rapidly cooled on ice and harvested by centrifugation at 1000 g for 20 min. The pellet was washed twice with ice-cold Mes buffer, pH 6.0, and resuspended in fresh Pi-starvation medium with 4% (w/v) glucose at a cell density of 25 mg of cells (wet weight) per ml. After 30 min of incubation, KH2PO4 was added to the culture and samples were taken at the indicated time points. For monitoring growth recovery after re-addition of Pi, the Pi-starved cells were resuspended in fresh Pi-starvation medium supplemented with KH2PO4 at an initial D600 of 0.1 and growth was followed by measuring D600 at the indicated time points. For the localization studies, cells were grown until mid-exponential phase in ScD and then starved of Pi and/or glucose or treated with 200 nM rapamycin for 6–8 h unless indicated otherwise. All incubations were done at 30 °C under continuous shaking.

Sampling, extraction and determination of trehalase activity

Samples of 50 mg of cells (wet weight) were taken at the indicated time points. Cells were rapidly cooled by addition of ice-cold water, centrifuged and resuspended in 500 μl of 25 mM Mes buffer pH 7, and 50 μM CaCl2, for extraction. Crude cell extracts were prepared as described previously [20] and dialysed (BRL microdialysis system) against 25 mM Mes buffer, pH 7, with 50 μM CaCl2 at 4 °C. Trehalase activity in dialysed cell extracts was determined as described previously [20]. The specific activity is expressed as nmol of glucose liberated/min per mg of protein. The total amount of protein in the samples was determined using a standard method [27].

Pi-consumption assay

After addition of 1 mM KH2PO4 to Pi-starved cells, 1-ml samples were taken and cells were rapidly pelleted at 4 °C. Pi levels in the supernatants were determined with the Fiske–Subbarow colorimetric method for the determination of Pi [28].

RNA extraction and Northern blot analysis

Samples of 120 mg of cells (wet weight) were taken at the indicated time points and rapidly cooled by addition of ice-cold water. The cells were pelleted and washed once with ice-cold water and stored at −80 °C. Isolation of total RNA was performed as described previously [20]. Probes were labelled with α-[32P]dCTP using the High Prime kit (Boehringer Mannheim). Northern blots were made by separation of total RNA on gels containing 1% (w/v) agarose in 50 mM boric acid, 1 mM sodium citrate, 5 mM NaOH, pH 7.5, and 1% formaldehyde. Subsequently, RNA was transferred by capillary blotting to a Hybond-N membrane (Amersham) using 10× SCC buffer (1.5 M NaCl and 0.15 M sodium citrate, pH 7.0). The filters were hybridized with the 32P-labelled probes consisting of fragments of the desired coding region. The blots were analysed using Fujix BAS-1000 PhosphorImager technology.

Fluorescence microscopy

Cells expressing EGFP-fusion constructs were grown as described above and used directly for microscopy without fixation. Cells were viewed using a Leica DM4000B fluorescence microscope. Pictures were taken with a Leica DFC420C camera using the Leica Application Suite software. For FM4-64 ([N-(3-triethylammoniumpropyl)-4-(p-diethylamino phenylhexatrienyl) pyridinium dibromide; Molecular Probes], which specifically stains the vacuolar membrane, strains overexpressing EGFP-fused Pho87 and Pho90 were Pi-starved for 6 h. The cells were stained with FM4-64 dye for 20 min at 4 °C and incubated at room temperature (20 °C) for 60 min to induce transport of FM4-64 via endocytotic intermediates to the vacuolar membrane and fluorescence of both the FM4-64 and the EGFP fusions was examined. Overlaying of images was performed with ImageJ.

Reproducibility of the results

All experiments were performed at least three times. For growth curves, trehalase and Pi-consumption experiments, representative Figures are shown. The absolute values are somewhat variable between different experiments, but the relative differences observed between separate experiments and strains are highly reproducible. For Northern blot experiments, representative blots from three independent experiments are shown.

RESULTS

The deletion of PHO4 shifts rapid Pi -signalling control from Pho84 to Pho87

Recent studies have demonstrated that Pho84 is the major Pi transporter in S. cerevisiae involved in rapid Pi signalling [22,23], as the pho84Δ strain is unable to rapidly reverse Pi-starvation responses. However, we previously observed rapid Pi signalling in strains deleted of PHO4 or PHO81 [19], both of which do not display expression of PHO84. Thus either expression of PHO84 below detection levels is sufficient to sustain Pi signalling or another Pi transporter takes over the function of Pho84 in the absence of Pho4 activity. To investigate this in more detail, the individual Pi transporters were deleted in a pho4Δ background. Upon re-addition of KH2PO4 to Pi-starved cultures, we observed that additional deletion of PHO4 not only restored Pi signalling in pho84Δ cells, but it also shifted the Pi-signalling control towards Pho87. The latter is illustrated by the absence of trehalase activation in a pho4Δ pho87Δ strain (Figure 1A), which nicely correlated with an inability to rapidly consume measurable amounts of Pi (Figure 1B). Rapid Pi signalling functions in part to quickly restart growth of the yeast cell, which is highlighted further by the rapid induction of ribosomal protein genes and the repression of the stress-responsive genes (Supplementary Figure S1A at http://www.BiochemJ.org/bj/434/bj4340243add.htm). Consequently, we observed that the pho84Δ and the pho4Δ pho87Δ mutants displayed retardation in growth resumption after addition of phosphate to Pi-starved cells when compared with the wild-type strain and the other mutants (Supplementary Figure S1B). This correlates well with the delayed induction of ribosomal protein genes and the delayed inhibition of stress-responsive genes in these strains (Supplementary Figure S1A). Notably, the presence of the other low-affinity Pi transporter, i.e. Pho90, does not substitute for Pho87 when it comes to Pi signalling in the pho4Δ strain (Figure 1), indicating that the two low-affinity Pi transporters are non-redundant. Deletion of PHO91 in a pho4Δ stain also did not affect rapid Pi signalling under these conditions (results not shown). Pho91 was not investigated further as this low-affinity Pi transporter was shown to mediate Pi transport across the vacuolar membrane [6]. Together, the results described above indicate that specifically the Pho87 Pi transporter mediates Pi signalling in the absence of a functional Pho4 transcription factor.

Figure 1 The low-affinity Pi transporter Pho87 is essential for rapid Pi signalling in cells lacking the transcriptional regulator Pho4

The wild-type strain (□) and the mutants pho84Δ (△), pho4Δ (▲), pho4Δ pho84Δ (◆), pho4Δ pho87Δ (■) and pho4Δ pho90Δ (◇) were grown to mid-exponential phase in ScD medium after which they were transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH2PO4 and (B) Pi consumption after re-addition of 1 mM KH2PO4 to the Pi-starved cells. spec. act., specific activity.

One possibility would be that Pho4 is somehow involved in transcriptional repression of PHO87. However, previous studies have already indicated that transcription of the low-affinity Pi transporters is independent of both Pi concentration and Pho4 activity [9]. This was further confirmed in the present study, since there was no significant alteration in the expression of either PHO87 or PHO90 when wild-type, pho84Δ or pho4Δ cells were starved of Pi, this being in contrast with the expression of PHO84 and PHO89, encoding the high-affinity Pi-transport system (Supplementary Figure S1C). Altogether, these results imply that, under Pi-limiting conditions, wild-type yeast cells down-regulate the low-affinity Pi transporter Pho87 and turn to the high-affinity Pho84 Pi transporter to signal and take up the available Pi. Both processes depend on the activation of the Pho4 transcription factor, which directly induces PHO84 expression and results in the inactivation of Pho87 at a post-transcriptional level.

Spl2 mediates Pi-starvation-induced vacuolar targeting of Pho87, but not of Pho90

In yeast, several plasma membrane nutrient transporters are inactivated and targeted to the vacuole in response to changes in the availability of nutrients [2932]. To determine whether the low-affinity Pi transporters Pho87 and Pho90 are subject to nutrient limitation-induced vacuolar targeting as well and to establish a role of Pho4 in this process, we set out to compare the intracellular localization of the EGFP-tagged Pho87 and Pho90 proteins under different nutritional conditions in the wild-type strain and in the pho4Δ mutant. For this, we opted to use the strains described previously [6], where chromosomal N-terminal EGFP-fusion genes of PHO87 and PHO90 were expressed by the TEF promoter. As shown in Figure 2(A), Pho87 was localized at the plasma membrane of wild-type cells grown on ScD medium, but this low-affinity transporter was endocytosed and targeted to the vacuole when cells encountered Pi starvation. This was additionally confirmed by the deletion of END3, encoding an essential component for endocytosis, which prevented the internalization of Pho87 and, as a consequence, led to the stabilization of this Pi transporter at the plasma membrane under Pi-starving conditions. Furthermore, the process could be monitored in wild-type cells when these were stained with FM4-64, a lipophilic dye that is endocytosed and used to mark endosomes and the vacuolar membrane (Supplementary Figure S2A at http://www.BiochemJ.org/bj/434/bj4340243add.htm). Intriguingly, the Pi-starvation-induced endocytosis and vacuolar targeting of Pho87 was prevented by the deletion of PHO4 (Figure 2A) as well as by the deletion of the Pho4-regulated gene SPL2 (Figure 2A and Supplementary Figure S2A). The latter was previously identified to encode a negative regulator of low-affinity Pi transport [24]. Next, we analysed Pi-starvation-induced endocytosis and vacuolar targeting of Pho90 in the wild-type and the pho4Δ, spl2Δ and end3Δ mutants using the PTEF-EGFP-PHO90 construct. In contrast with the Pho87 Pi transporter, endocytosis of Pho90 was only prevented in the mutant strain lacking End3 and not in the strains lacking Pho4 or Spl2, indicating that the Pi-starvation-induced turnover of Pho90 occurred independently of the latter (Figure 2B and Supplementary Figure S2B). We then examined a possible involvement of the protein kinase Rim15, another effector of the Pho85–Pho80 kinase complex [19,33]. However, this effector did not seem to play a role in the control of the low-affinity transporters, since the deletion of RIM15 did not affect the removal of Pho87 or Pho90 from the plasma membrane during Pi starvation (Figure 2).

Figure 2 Spl2 mediates the vacuolar targeting of Pho87, but not of Pho90, under Pi-starving conditions

Cells carrying a chromosomal PTEF-EGFP-PHO87 (A), PTEF-EGFP-PHO87Δ375N (A), PTEF-EGFP-PHO90 (B) or a PTEF-EGFP-PHO90Δ375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6–8 h to Pi-starvation medium (-Pi). As indicated, the wild-type (WT) cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. More than 300 fluorescent cells were counted and >90% of the cells exhibited the phenotypes visualized in the Figure.

Recently, Spl2 was shown to interact with the N-terminal SPX domain of Pho87 and Pho90 [4]. This led us to analyse Pi-starvation-induced endocytosis and vacuolar targeting of Pho87Δ375N and Pho90Δ375N, representing Pho87 and Pho90 constructs lacking the N-terminal 375 amino acids. The endocytosis of Pho87 and Pho90 under Pi-starvation conditions was abrogated in the absence of their SPX domain (Figure 2). For Pho87, this observation fits with the proposed role of Spl2 as an inhibitor of low-affinity Pi transport. For Pho90, however, the Pi-starvation-induced turnover occurs independently of Spl2 and, therefore, our results suggest that this process requires the interaction of another factor with the SPX domain.

The SPX domain is essential for vacuolar targeting of Pho87 and Pho90 in response to glucose starvation and rapamycin treatment

It has been reported that the Pi-induced degradation of Pho84 is delayed in strains with reduced PKA activity [22,34]. As PKA is part of a nutrient-responsive network, which also comprises the protein kinases Sch9, Rim15 and TORC1, we investigated whether other nutritional cues, besides Pi, affected the internalization and subsequent vacuolar targeting of Pho87 and Pho90. First, to test the effect of carbon-source availability, cells were transferred from ScD to Sc medium lacking a carbon source for 6–8 h. Under these conditions, Pho87 and Pho90 were also endocytosed and targeted to the vacuole, and this process could not be prevented by deletion of PHO4 or SPL2 (Figure 3). Interestingly, shifting the cells from ScD medium to Sc medium containing galactose or glycerol already induced endocytosis of both low-affinity Pi transporters (results not shown), letting us conclude that a down-shift in the quality of the carbon source is sufficient to initiate the process. Since Rim15 acts as a nutritional integrator in S. cerevisiae, we also analysed the effect of a RIM15 deletion on endocytosis and vacuolar targeting of both Pi transporters but, similar to Pi starvation, the Rim15 kinase appeared not to be required (Figure 3).

Figure 3 The SPX domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi transporters during glucose-starvation conditions and after rapamycin treatment

Cells carrying a chromosomal PTEF-EGFP-PHO87 (A), PTEF-EGFP-PHO87Δ375N (A), PTEF-EGFP-PHO90 (B) or a PTEF-EGFP-PHO90Δ375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6–8 h to medium without glucose (-D). In addition, the localization of both Pi transporters was monitored after treatment of exponentially grown cells for 6–8 h with 200 nM rapamycin (Rap). As indicated, the wild-type (WT) cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. More than 300 fluorescent cells were counted and >90% of the cells exhibited the phenotypes as visualized in the Figure.

Next, we challenged the cells with the TORC1-inhibiting drug rapamycin, a condition known to mimic amino acid and nitrogen starvation and to induce vacuolar targeting of different permeases in yeast, such as Gap1 and Tat2 [35,36]. As shown in Figure 3, treatment of yeast cells with rapamycin also triggered endocytosis and vacuolar targeting of Pho87 and Pho90 and this was again independent of Pho4, Spl2 and Rim15. Similar results were also obtained for nitrogen starvation (results not shown).

Since the SPX domain is essential for endocytosis and vacuolar targeting of Pho87 and Pho90 under Pi-starvation conditions, we wondered whether this domain also directs endocytosis upon glucose starvation and rapamycin treatment. Therefore we analysed the internalization of the Pho87Δ375N and Pho90Δ375N constructs, which revealed that the SPX domain is indeed required to allow endocytosis of the two low-affinity Pi transporters under both conditions (Figure 3). Thus, despite the difference in the underlying control mechanisms and proteins involved, the internalization of the low-affinity Pi transporters during Pi starvation, glucose starvation or rapamycin treatment is based on the same principle, i.e. interactions of endocytosis-inducing proteins with the SPX domain of the transporter.

The Pho90 low-affinity Pi transporter is required for proper growth under high Pi conditions in the absence of a high-affinity Pi-transport system

The results described above focussed on the role and turnover of the low-affinity Pi transporters under starvation conditions. In the next section, the physiological role of the low-affinity Pi transporters was investigated when sufficient amounts of Pi are present. Therefore we evaluated the contribution of Pho87 and Pho90 to sustain growth of mutant strains lacking a functional high-affinity Pi-transport system, in relation to increasing levels of extracellular Pi. In these mutants, the high-affinity transport system was inactivated, either by deletion of PHO4, thereby inhibiting the transcription of PHO84 and PHO89, or by the combined deletion of PHO84 and PHO89. The absence of a high-affinity Pi-transport system results in a clear growth defect once the extracellular Pi levels fall below 250 μM (Figure 4A). We then additionally deleted either Pho87 or Pho90 in order to determine in more detail the contributions of each of the low-affinity Pi transporters to sustain growth at different extracellular Pi concentrations. The pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strains already displayed a clear growth defect once the extracellular Pi concentrations fall below 5 mM, whereas the pho4Δ pho87Δ and the pho84Δ pho89Δ pho87Δ strains displayed a growth defect only at Pi concentrations of 500 μM and below (Figure 4A). This indicates that mainly the Pho90 low-affinity Pi transporter is important when sufficient amounts of extracellular Pi are available.

Figure 4 The Pho90 Pi transporter is required under high Pi conditions in the absence of a high-affinity Pi-transport system

(A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strains) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strains) at the plasma membrane was compared with the growth of strains only containing both low-affinity Pi transporters (the pho4Δ and the pho84Δ pho89Δ strains). Cells were grown in ScD medium and 10-fold serial dilutions were spotted on to Pi-starvation medium containing a specific amount of Pi. Pictures were taken after 2–3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and 10-fold serial dilutions were spotted on to YPD, YPD containing an additional 10 mM Pi and/or 5mM AsO4. Pictures were taken after 3–5 days. WT, wild-type.

These results were additionally confirmed when the growth of the different strains was further analysed on rich YPD medium and in the presence of the toxic phosphate analogue arsenate. On YPD medium, specifically the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strains exhibit growth defects when compared with the other strains examined. Consistent with the findings above, these defects can be suppressed by the addition of 10 mM Pi, indicating again the importance of Pho90 for Pi uptake to sustain growth (Figure 4B). Moreover, when arsenate was added to the medium, an increased resistance to this toxic phosphate analogue was observed for both the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strains (Figure 4B).

DISCUSSION

The low-affinity Pi transporters Pho87 and Pho90 are controlled by endocytosis in response to nutrient starvation

Our results concerning the rapid phosphate signalling indicated that Pho4 inhibits the function of the low-affinity Pi transporter Pho87. This inhibition appeared to be independent of PHO87 transcription, which, in agreement with previous findings [9], is not regulated by Pi concentration or by major components of the PHO pathway, such as Pho4 and Pho84. This indicated that the regulation of Pho87 in its functioning in Pi availability must occur primarily at a post-transcriptional level and, consistently, we demonstrated that both the Pho87 and Pho90 transporters are targeted to the vacuole when yeast cells are starved of phosphate. It was previously reported that this process probably involved ubiquitination-induced endocytosis [37], which is in agreement with our results showing a complete stabilization of Pho87 and Pho90 at the plasma membrane in Pi-starved end3Δ cells. Interestingly, the vacuolar targeting is impaired specifically for Pho87 in cells deleted for PHO4 or SPL2, whereas Pho90 is still routed to the vacuole in the pho4Δ strain. As the Pho87Δ375N protein, lacking its SPX domain, also failed to be targeted to the vacuole in the absence of phosphate, our results suggest a role for Spl2 in mediating endocytosis of Pho87 via interaction with its SPX domain [4]. Interestingly, the Pho90Δ375N protein displayed a similar localization pattern, indicating that, also for Pho90, the SPX domain is required for its endocytosis and vacuolar targeting under phosphate starvation, although in this case the process is largely independent of the Pho4/Spl2 branch of the PHO pathway. Hence it can be concluded that two independent mechanisms regulate the selective vacuolar targeting of the Pho87 and Pho90 low-affinity phosphate transporters in response to depletion of phosphate from the medium, both of which are absolutely dependent on interactions with theN-terminal 375 amino acids of these proteins.

Our data on endocytosis and vacuolar targeting of the low-affinity Pi transporters clarify the results obtained in the context of phosphate signalling. Since in a wild-type strain both Pho87 and Pho90 are subject to vacuolar targeting during phosphate starvation, rapid phosphate consumption and signalling largely depends on the high-affinity transporter Pho84 (Figure 5). However, in a strain lacking Pho4, transcription of PHO84 is absent, rendering the cells unable to signal phosphate availability via the Pho84 transporter. However, in this strain, specifically Pho87 remains stabilized at the plasma membrane under phosphate starvation, while Pho90 is still undergoing vacuolar targeting, hence rapid phosphate consumption and signalling becomes, indeed, strictly dependent on this low-affinity Pi transporter (Figure 5).

Figure 5 Regulation of yeast Pi transporters during Pi starvation

When Pi becomes limiting, the activity of the Pho85–Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, whereas Pho90 is targeted to the vacuole in an Spl2-independent manner. Although not depicted here, we have also demonstrated that, under acute glucose starvation and after the addition of rapamycin, the low-affinity Pi transporters Pho87 and Pho90 are both targeted to the vacuole in an Spl2-independent way.

We also demonstrate that, besides control by Pi availability, other nutrients are able to regulate the low-affinity Pi-transporter turnover. This was documented for glucose starvation and rapamycin treatment, the latter being a condition that mimics starvation of nitrogen and amino acids [36]. Both conditions were found to affect both Pho87 and Pho90, and we confirm that this turnover is also driven by endocytosis [22,37,38]. Although the vacuolar targeting of both Pi transporters under these conditions does not involve Pho4/Spl2, the SPX domains of Pho87 and Pho90 are again essential for this process. This strengthens our hypothesis that other SPX-binding factors are involved in mediating endocytosis and vacuolar targeting. Furthermore, the results indicate that both glucose starvation and rapamycin treatment induce a general vacuolar sorting response that overrules the Pi-specific feedback loops. This may allow the cell to adjust Pi uptake not only as a function of the external Pi concentration, but also to balance the Pi uptake with respect to other metabolic processes, thereby preventing an overflow of internal Pi pools.

As our present study indicates that both Spl2 and the SPX domain of Pho87 are required for Pi-starvation-induced endocytosis of Pho87, these results fit well with the previously reported interaction of Spl2 with the SPX domain of this Pi transporter [4]. In the same study, however, a similar interaction of Spl2 was observed with the SPX domain of Pho90, whereas we did not observe a necessity for Spl2 in the endocytosis of Pho90 upon Pi starvation, although the SPX domain of the latter still seemed absolutely required. These results seem contradictory, but one has to keep in mind the differences in experimental approaches in the present study compared with the previous one [4]. The negative interaction between Spl2 and Pho90, as described in [4], was seen in strains overexpressing PHO90 and/or SPL2 on rich YPD medium, which contains intermediate levels of Pi. In contrast, in the present study, effects were observed upon deletion of SPL2 in Pi-starvation medium. In addition, our results obtained under different nutritional conditions, as described above, indicate that other factors, besides Spl2, exist that mediate endocytosis of both Pho90 and Pho87. Consequently, overexpression of SPL2 may generate dominant effects overruling the regulation of the Pi transporters by other factors, whereas deletion of SPL2 makes the regulation of Pho87 and Pho90 exclusively dependent on these factors. It is therefore very likely that Spl2 mediates the regulation of Pho90 activity under certain physiological conditions not addressed in the present study. In this respect, we note that, although a transcriptional regulation of SPL2 in response to Pi-levels is already documented [10,24], other modes of regulation of Spl2 function may exist which respond to different environmental cues.

Non-redundant functions for the Pho87 and Pho90 low-affinity Pi transporters

In the present study, we have unveiled different situations in which either the Pho87 or the Pho90 Pi transporter is required for signalling and/or optimal growth. Indeed, we have shown that, in a pho4Δ background, Pi signalling and rapid growth recovery from Pi starvation depends specifically on the Pho87 transporter, whereas, under conditions where sufficient amounts of Pi are available, Pho90 is the most important low-affinity Pi transporter. These transporter-specific requirements signify that Pho87 and Pho90 represent non-redundant Pi transporters that are tuned by the integration of multiple nutrient signalling mechanisms in order to adjust Pi-transport capacity to the general nutritional status of the environment.

AUTHOR CONTRIBUTION

Ruben Ghillebert, Erwin Swinnen, Pepjin De Snijder, Bart Smets and Joris Winderickx conceived and designed the experiments and analysed the data. Ruben Ghillebert and Erwin Swinnen performed the experiments. Ruben Ghillebert, Erwin Swinnen and Joris Winderickx wrote the paper.

FUNDING

This work was funded by the K.U. Leuven Research Fund (K.U. Leuven-BOF); the Fund for Scientific Research-Flanders (FWO-Vlaanderen) [grant number G.0430.08]; and IWT Vlaanderen (Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen) (fellowships granted to R.G. and P.D.S.).

Acknowledgments

We thank Professor Dr H.C. Hürlimann (Institute of Plant Sciences, ETH Zurich, Zurich, Switzerland) for providing the EGFP-tagged low-affinity Pi-transporter strains.

Abbreviations: CKI, cyclin-dependent kinase inhibitor; EGFP, enhanced green fluorescent protein; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylamino phenylhexatrienyl) pyridinium dibromide; PKA, protein kinase A; ScD, Sc (synthetic complete) minimal medium containing 0.5% (NH4)2SO4 and 1.9 g/1 yeast nitrogen base (without amino acids), supplemented with all amino acids/nucleotides and 2% (w/v) glucose; TORC1, target of rapamycin complex 1

References

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