PHM6 and PHM7 genes are essential for phosphate surplus in the cells of Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae are capable for phosphate surplus: the increased uptake of phosphate (Pi) and accumulation of inorganic polyphosphate (polyP) occur when the cells after Pi limitation were cultivated in a medium supplemented with Pi. We demonstrated that single knockout mutations in the PHO84, PHO87, and PHO89 genes encoding plasma membrane phosphate transporters suppressed the Pi uptake and polyP accumulation under phosphate surplus at nitrogen starvation. The knockout strains in the PHM6 and PHM7 genes encoding unannotated PHO-proteins showed decreased polyP accumulation under Pi surplus both at nitrogen starvation and in complete YPD medium. This is due to the suppression of Pi uptake in the cells of these mutant strains. We speculate that Pi transporters of plasma membrane, and Phm6 and Phm7 proteins function in concert providing increased Pi uptake at phosphate surplus conditions.


Introduction
The accumulation of phosphate reserves as inorganic polyphosphate (polyP), a linear polymer containing from several to hundreds of phosphate residues, is a common strategy for maintaining phosphorus homeostasis in microbial cells (Rao et al. 2009;Zhang et al. 2022). The microbial species belonging to various systematic groups demonstrates the phenomenon of phosphate surplus: if the cells after phosphate (Pi) starvation were cultivated in a Pi-containing medium, they accumulated higher polyP level compared to the cells grown in a Pi-sufficient medium (Kulaev and Vagabov 1983). The yeast Saccharomyces cerevisiae accumulates high polyP level under phosphate surplus conditions (Kulaev and Vagabov 1983;Christ and Blank 2019). More than 300 genes are involved in the regulation of the PHO pathway in yeasts (Choi et al. 2017).
The key components of polyP accumulation in yeast cells are the VTC complex and phosphate transporters. In yeasts, the Vtc4 protein performs polyP synthesis (Hothorn et al. 2009). Vtc4 is a part of the Vacuole Transporter Chaperone (VTC) complex (Müller et al. 2002(Müller et al. , 2003. The VTC in the cells of S. cerevisiae may contain Vtc4/Vtc3/Vtc1 proteins or Vtc4/Vtc2/Vtc1 proteins; the first variant is localized in the vacuolar membrane, while the second variant was observed in the vacuolar membrane under Pi starvation, in the endoplasmic reticulum, and the nuclear envelope (Gerasimaite and Mayer 2016). Vtc2, Vtc3, and Vtc4 contain the SPX domains, which is responsible for the interaction with inositol pyrophosphate signaling molecules, whose concentrations change depending on the availability of Pi (Gerasimaite et al 2014(Gerasimaite et al , 2017Wild et al 2016). The Vtc5 also contains a SPX domain and participates in the regulation of polyP synthesis (Desfougères et al. 2016).
Yeast cells possess several phosphate transporters (Table 1). There are two low affinity H + /Pi symporters Pho87 and Pho90, high-affinity H + /Pi symporter Pho84, and highaffinity Na + /Pi symporter Pho89 in the plasma membrane (Mouillon and Persson 2006;Eskes et al. 2018). The lowaffinity phosphate/sodium symporter Pho91 localized in the vacuolar membrane is essential for the storage and mobilization of vacuolar polyP (Potapenko et al. 2018 Among the genes belonging to PHO pathway are PHM6 and PHM7. Their expression significantly increased in yeast cells grown under Pi limitation, and also was induced by treatment with the drugs inhibiting Cdc28 and Pho85 kinases (Ogawa et al. 2000). Phm7 protein is localized in plasma membrane and contains several transmembrane domains (Huh et al. 2003; The UniProt Consortium 2012). The need for the Phm7 protein for adaptation to toxic Mn 2+ concentration has been reported (Trilisenko et al. 2019). Its ortholog CaPhm7 in Candida albicans localized in the plasma membrane participates in drug resistance and filamentous growth (Jiang and Pan 2018). The cells lacking CaPHM7 are sensitive to SDS and ketoconazole but resistant to rapamycin and zinc (Jiang and Pan 2018). No data on the functions of Phm6 protein in S. cerevisiae are available. The possible role of Phm6 and Phm7 in phosphate metabolism remains unknown.
The aim of this study was to reveal the possible role of Phm6 and Phm7 of S. cerevisiae in Pi and polyP accumulation under Pi surplus conditions.

Strains and growth conditions
The S. cerevisiae wild-type (WT) strain BY4741 and the knockout mutant strains were obtained from the Dharmacon collection. Cells were cultivated in the full YPD medium containing 2% glucose, 2% peptone (Pronadisa, Madrid, Spain), and 1% yeast extract (Pronadisa, Madrid, Spain). This medium contained 5.5 mM Pi. The Pi-limited YPD prepared according to Rubin (Rubin 1973) was used for cultivation at Pi limitation. This medium contained 0.194 ± 0.004 mM Pi. The cells were grown at 28 °C and 145 rpm for the stationary growth stage, harvested by centrifugation at 5000 g for 10 min, and washed twice with sterile distilled water. The wet biomass quantity was normalized by weighting biomass samples standardized under centrifugation conditions applied in our previous studies, 5000 g for 15 min (Vagabov et al. 2000).

Pi surplus at non-growth conditions
Freshly harvested yeast cells (~ 55 mg wet biomass) were incubated in 0.75 mL of MiliQ water containing 100 mM glucose and 0.8-1 mM K 2 HPO 4 and supplemented or not with 5 mM MgSO 4 at 30 °C with shaking (850 rpm) in ThermoMixer (Eppendorf, Hamburg, Germany). After 15-45 min, the cells were centrifuged at 14,000 g for 3 min, and Pi was assayed in supernatants by the colorimetric method with malachite green (Andreeva et al. 2019). The precipitates of biomass were washed with MiliQ water three times at 14,000 g for 3 min and used for Pi and polyP extraction.

Pi surplus in YPD medium
To assess the effect of the PHM6 and PHM7 gene knockout on the phosphate surplus in YPD medium, the cells of the wt, Δphm6 and Δphm7 strains were grown in a Pi-limited YPD medium (0.194 mM Pi) for 24 h at 28 °C and 145 rpm. The biomass samples were separated by centrifugation at 5000 g for 15 min, washed twice with MiliQ water, and suspended in MiliQ water (1 g wet biomass/mL). Biomass samples were used to assay the Pi and polyP content in the cells grown in a phosphate-limited medium. To obtain the effect of phosphate surplus, the cells were transferred to the YPD

Pi and polyP extraction and assay
Acid soluble polyP was extracted from biomass with 0.5 M HClO 4 at 0 °C as described earlier (Vagabov et al. 2000). PolyP content in the extracts was estimated by phosphate releasing after 20-min treatment with 1 M HCl at 90 °C. The amount of acid insoluble polyP remaining in the biomass was estimated by the phosphate released after 20-min treatment of biomass samples with 1 M HClO 4 at 90 °C (Vagabov et al. 2000). Phosphate was measured as described earlier (Andreeva et al. 2019).
The experiments were performed in triplicate; the average values and their standard deviation calculated using Excel are presented.

Fluorescence microscopy
The S. cerevisiae GFP clones PHM6-GFP and PHM7-GFP derived from BY4741 strain were obtained from the Dharmacon collection. For fluorescence microscopy the cells of both strains were cultivated in full YPD medium, and in Pi-limited YPD medium and also incubated at Pi surplus (45 min incubation with 100 mM glucose, 1 mM K 2 HPO 4 and 5 mM MgSO 4 ). The cells were examined by fluorescent microscopy AXIO Imager A1 (Zeiss, Germany) with a filter set 56HE (Zeiss) at a wavelength of 480 nm (maximum excitation) and 512-630 (emission). An Axiocam 506 (Zeiss) was used to acquire images.

The effects of knockout mutations in the phosphate transporter genes, PHM6, and PHM7 on Pi and polyP accumulation in non-growth conditions
Earlier, we showed that the yeast cells pre-grown in Pilimited medium demonstrated increased Pi and polyP accumulation at nitrogen starvation, in the medium containing only glucose and Pi (Breus et al. 2012;Tomashevsky et al. 2021). Both processes were stimulated by Mg 2+ (Breus et al. 2012). During incubation in these conditions, no increase in cell density was observed even for 1-5 h, indicating that the cells did not have a complete budding cycle (Breus et al. 2012). We compared Pi and polyP accumulation at Pi surplus in non-growth conditions in the WT strain and strains with knockout mutations in the phosphate transporter genes, PHM6 and PHM7. To estimate the value of Pi accumulation we assayed the decrease in Pi concentration in the medium after incubation with the yeast cells. The lower this concentration, the more Pi is absorbed by the cells.
The cells pre-grown in the complete medium with 5.5 mM of Pi demonstrated negligible Pi accumulation (Supplementary Table 1). The cells of WT strain pre-cultivated in a Pi-limited medium containing 0.194 mM Pi, demonstrated increased Pi accumulation, especially in the presence of Mg 2+ (Table 2). Only Δpho91 strain was similar to the WT strain (Table 2). Both strains almost completely took up Pi from the medium within 45 min. The cells of the Δpho84 and Δpho90 strains showed low levels of Pi accumulation. The Pi accumulation in the cells of strains Δpho87, Δpho89, Δphm6, and Δphm7 strains was significantly suppressed in the presence of Mg 2+ and was not observed in the absence of Mg 2+ . Based on the data shown in Table 2 and Supplemental  Table 2, we calculated the rates of Pi uptake by the cells of the studied strains (Fig. 1). Even the cells of Δpho91 strain had the lower uptake rate compared to the WT strain.
We expected the decrease in Pi accumulation at phosphate surplus in the cells with knockouts in the genes encoding the plasma membrane Pi transporters. Surprisingly, a single knockout in PHO84, PHO87, PHO89, or PHO90 was enough to almost completely suppressing of the Pi accumulation at nitrogen starvation. The same effect was observed with knocked out PHM6 or PHM7 genes. Consequently, Phm6 and Phm7, which are not Pi transporters, participate in increased Pi uptake at phosphate surplus conditions.
We have assayed the polyP content in the cells both pre-cultivated in Pi limited medium and in the cells subjected to phosphate surplus in non-growth conditions. The Table 2 Pi concentration in the medium after 45 min incubation with the cells of S. cerevisiae pre-grown in phosphate-limited YPD medium (0.194 ± 0.004 mM Pi) The cells were incubated in water containing 100 mM glucose and 1 mM K 2 HPO 4 (-MgSO 4 ) or in water containing 100 mM glucose, 1 mM K 2 HPO 4 , and 5 mM MgSO 4 (+ MgSO 4 ). Control-the medium was incubated without cells, and Pi concentration was measured with the same assay method cells of all strains grown in a Pi-limited medium had the low content of both acid soluble (Fig. 2) and acid insoluble polyP (Supplemental Table 3) similar to other yeast strains (Vagabov et al. 2000). The content of Pi in the cells of all strains under study was still unchanged at phosphate surplus (Supplemental Table 3). The content of acid insoluble polyP after 45-min incubation only slightly increased in the cells of some strains (Supplement Table 3). Earlier it was shown, that at phosphate surplus the amount of the low-molecular acid soluble polyP increased for the first hours, whereas the amount of high-molecular acid insoluble polyP increased latter (Vagabov et al. 2000). Hence, we have focused attention on the acid-soluble polyP. The accumulation of acid soluble polyP depended on the level of Pi accumulation under our experimental conditions (Fig. 2). The content of acid soluble polyP in the cells of WT strain and the Δpho90 and Δpho91 strains considerably increased after 45-min incubation compared to the Pi-limited cells (Fig. 2). In the Δpho91 strain, the amount of acid soluble polyP was even higher than in the WT strain. Probably, this is due to the peculiarities of polyP and Pi homeostasis in vacuoles when Pi export from the vacuoles is impaired. The Δpho84 strain demonstrated an increase in the content of acid soluble polyP; however, this increase was significantly lower compared to the WT strain. The strains Δpho87, Δpho89, Δphm6 and Δphm7 showed relatively small increase in the content of acid-soluble polyP compared to the cells pre-grown in Pilimited medium. Thus, the impaired Pi uptake in the cells of Δpho84, Δpho87, Δpho89, Δphm6 and Δphm7 strains leads to suppression of polyP accumulation under phosphate surplus conditions. The PHM6 or PHM gene knockout results in the impairment of both Pi uptake and polyP accumulation at phosphate surplus in non-growth conditions.

Expression Phm6-GFP and Phm7-GFP
Using the strains containing PHM6 and PHM7 fused with GFP, we tested Phm6 and Phm7 expression by fluorescence microscopy. The cells grown in complete YPD medium had negligible expression of both proteins, while the cells grown in Pi -limited YPD medium and the cells exposed to Pi surplus demonstrated increased expression of Phm6 and Phm7 ( Supplementary Fig. 1). These data are consistent with DNA Microarray Analysis (Ogawa et al. 2000). Fluorescence microscopy indicate the association of Phm6 and Phm7 with the cell periphery. These data are consistent with the assumption that these proteins play an important role in increased Pi uptake at phosphate surplus.

The Δphm6 and Δphm7 strains have decreased polyP level under growth at Pi surplus
We have tested the effects of the PHM6 or PHM7 gene knockout on polyP accumulation under Pi surplus in the complete YPD medium. The cells of WT strain, Δphm6, and Δphm7 strains were pre-grown in Pi-limited medium for 24 h, and after them cultivated in YPD medium with 16 mM Pi for 2 h. The cells of these strains grown in Pi-limited medium showed no difference in biomass production (data not shown) and in the Pi and polyP levels (Supplemental Table 4). After cultivation at Pi surplus conditions for 2 h, the level of acid soluble polyP in the cells of WT strain was threefold higher than in the cells of both mutant strains, while the level of acid insoluble polyP was twofold higher (Fig. 3). Therefore, the knockout mutation in PHM6 or PHM7 genes results in impairment of the polyP accumulation at Pi surplus in complete culture medium. We suggest, that this is due to the suppression of increased Pi transport. The effects of knockouts in PHM6 or PHM7 genes on polyP level were similar in a complete medium and in the medium containing glucose, Pi, and Mg 2+ .
In conclusion, we suggest that one of the functions of Phm6 and Phm7 proteins is the participation in increased phosphate uptake by yeast cells previously subjected to phosphate limitation and then subjected to phosphate surplus. The data on the effects of single mutations in the PHO84, PHO87, and PHO89 genes on Pi and polyP accumulation allow us to speculate that the Pi transporters of the cytoplasmic membrane, Pho84, Pho87 and Pho89, work in concert under phosphate surplus conditions, providing increase of Pi uptake. The PHM6 and PHM7 genes encoding transmembrane proteins are probably essential for the interaction between the above Pi transporters.