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Saccharomyces cerevisiae has over the years provided a model for studies of how a cell makes a coordinated response in adapting to environmental changes in phosphate levels (7, 11). This and other microorganisms have evolved complex mechanisms to efficiently take up this essential nutrient, which is often present in low amounts in the environment. When the cells meet a limitation in external phosphate, a high-affinity transport system with a K_m for external phosphate of 0.5 to 10 µM is mobilized (3, 8). Of the proteins responsible for the high-affinity uptake, one is an H⁺-coupled phosphate cotransporter encoded by the PHO84 gene (3). The activity of the Pho84p transporter has been shown to be regulated by the external phosphate level through expression of the gene, sorting of the synthesized protein to the plasma membrane, and degradation by rerouting of the protein to the vacuole (9, 12). The other high-affinity phosphate transporter is encoded by the PHO89 gene (8). The Pho89p transporter is largely inactive at the pH optimum for Pho84p-mediated transport, suggesting that this transporter has a complementary role in cellular phosphate acquisition. In this study, we have characterized the regulation and activity of the Pho89p transporter by use of mutants lacking either the Pho84p or the Pho89p transporter.

The S. cerevisiae strains used were MB191 (MATa pho3-1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (3), MB192 (MATa pho3-1 pho84::HIS3 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (3), PAM1 (MATa pho3-1 pho89::TRP1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (8), and PAM2 (MATa pho3-1 pho84::HIS3 pho89::TRP1 ade2 leu2-3,112 his3-532 trp1-289 ura3-1,2 can1) (8). Cells were routinely grown in shaking Erlenmayer flasks at 30°C in low-phosphate (LP_i) medium (5), pH 4.5, to an optical density at 600 nm (OD₆₀₀) ranging from 0.1 to 4.5. Cells were harvested by centrifugation at 2,300 × g for 10 min and washed either once with 25 mM Tris-succinate (for P_i uptake assays), at a different pH for each experiment, or twice with ice-cold bidistilled water (for ³¹P nuclear magnetic resonance [NMR] measurements). The supernatants were subjected to phosphate concentration measurements spectrophotometrically essentially as described previously (10).

Phosphate uptake in pho84 cells was assayed by the addition of 1-µl volumes of [³²P]orthophosphate (0.18 Ci/µmol; 1 mCi = 37 MBq; Amersham-Pharmacia Biotech) to 30-µl aliquots containing (each) 3 mg (wet weight) of cells suspended in 25 mM Tris-succinate buffer, pH 8.5, supplemented with 3% glucose, to a final concentration of 50 µM P_i, in the presence of 25 mM NaCl. The suspension was immediately mixed and incubated at 25°C. P_i transport was terminated at given time intervals, in the range of 0.5 to 15 min, by adding 1 ml of ice-cold Tris-succinate dilution buffer. The sample was filtered immediately, the filter (Whatman GF/F) was washed once with the same ice-cold buffer, and the radioactivity retained on the filters was determined by liquid scintillation spectrometry. Phosphate uptake in pho89 cells and in pho84 pho89 cells was assayed under the same condition used for the pho84 cells, with the exception that the pH and the P_i concentration were 4.5 and 0.22 mM, respectively.

All NMR experiments were conducted on a Varian INOVA 500-MHz spectrometer. Wild-type, pho84, and pho89 cells were harvested at OD₆₀₀ values of 0.5, 1.5, and 3.0. Samples analyzed consisted of 3.0-ml aliquots of cell suspensions (0.5 g [wet weight] of cells/ml) in 25 mM Tris-succinate buffer, pH 4.5. A broad-band probe designed for 10-mm-sample tubes was used. The spectral width was 7,267 Hz. Phosphoric acid (85%), 0 ppm, was used as an external reference. The pulse delay was 2 s, and 1,024 scans of 8,192 complex data points were collected during an experimental time range of approximately 40 min. The 90°C excitation pulse length was determined to be 22 µs. No deuterium frequency lock was used during the experiments. The relative contributions of different ³¹P-containing molecules were derived from the corresponding peak area intensities in the ³¹P NMR spectra. The assignment of the ³¹P NMR peaks of intra- and extracellular orthophosphate and nonterminal P_i of polyphosphate were obtained from the literature (4).

Previous studies on P_i transporter gene expression in S. cerevisiae have shown that the PHO84 and the PHO89 transcripts are induced under P_i-deficient conditions (3, 8). The induction of the PHO84 transcript and synthesis of the transporter require that the concentration of external P_i be lower than 100 µM (9, 12).

To further analyze the functional expression of the Pho84p and the Pho89p phosphate transporters in cells grown in LP_i medium, we compared the phosphate transport properties of the three mutant strains (pho84, pho89, and pho84 pho89 mutants). In agreement with the behavior of wild-type cells (9), mutant cells lacking the Pho89p transporter revealed an activation of [³²P]phosphate uptake at pH 4.5 when measured at an OD₆₀₀ of 0.5, corresponding to a situation when the external P_i concentration had decreased from the initial concentration of 180 µM to 40 µM. As in the case of wild-type cells (9), the mutant reached its maximum transport activity (12.5 µmol · g of cells¹ · min¹) at an OD₆₀₀ of close to 2 when the external P_i was close to exhausted (Fig. 1A). Continued growth of the pho89 cells resulted in a rapid inactivation of the high-affinity [³²P]phosphate transport. In order to investigate the contribution of the low-affinity P_i transport system in pho89 cells assayed at pH 4.5, the pho84 pho89 double-disruptant strain was used (Fig. 1A). The [³²P]phosphate transport catalyzed by these cells was at least 20-fold lower than that of the pho89 cells over the OD₆₀₀ range studied. In contrast to the pho89 cells, which at an OD₆₀₀ of 0.5 had consumed about 75% of the available P_i in the growth medium, the double disruptant grown and assayed under identical conditions catalyzed a transient efflux of intracellular P_i, resulting in a twofold increase of the external P_i concentration compared to that originally contained in the growth medium. The double-disruptant cells grown to OD₆₀₀ values exceeding 0.5, however, regained the ability to take up the excreted P_i (Fig. 1A). Thus, it appears that the [³²P]phosphate transport activity and the rapid consumption of extracellular P_i observed in pho89 cells is catalyzed by the high-affinity Pho84p transporter without a significant contribution by the low-affinity transport system.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) [32P]orthophosphate uptake catalyzed by pho89 () and pho84 pho89 () cells at pH 4.5. Cells were grown in LPi medium and collected when the OD600 reached the value indicated. The supernatants of pho89 cells () and of pho84 pho89 cells () were used for phosphate determination. (B) [32P]orthophosphate uptake catalyzed by pho84 cells at pH 8.5 in the presence () or absence () of Na+. Cells were grown as described for panel A. The supernatant of the cells was used for phosphate determination (). (C and D) Intracellular levels of inorganic phosphate and polyphosphate, respectively, in wild-type (black bars), pho84 (shaded bars), and pho89 (white bars) cells were measured by 31P NMR.

In order to investigate whether the Pho89p transport activity is subjected to regulation by external P_i, LP_i-grown pho84 cells were assayed for [³²P]phosphate uptake at pH 8.5 (Fig. 1B). Although high-affinity P_i transport in both pho89 and pho84 cells revealed a pronounced OD₆₀₀ dependence, activation of the Pho89p transporter occurred at an earlier stage of the growth phase, reaching its maximum (0.06 µmol · g cells¹ · min¹) at an OD₆₀₀ of 0.5, at which point the pho89 cells do not catalyze a significant [³²P]phosphate uptake (the level was 14-fold lower) (data not shown). In agreement with a previous proposal that the Pho89p catalyzes a cation-dependent transport (8), the activity of the Pho89p transporter expressed in pho84 cells in the absence of Na⁺ was almost completely abolished (Fig. 1B). In contrast to the activation of the Pho84p transporter in pho89 cells at pH 4.5, which was paralleled by a lowered external P_i concentration, pho84 cells catalyzed a rapid initial P_i efflux at OD₆₀₀ values lower than 0.5, resulting in a twofold increase in external P_i, after which these cells, like the double-disruptant cells, regained the ability to take up external P_i.

Given the high degree of similarity in functional expression and external P_i dependence of Pho84p in the wild-type and the pho89 cells and the difference observed in the case of pho84 cells, it was likely that activation of the two transporters would be reflected by an altered cellular level of P_i. LP_i-grown wild-type, pho84 and pho89 cells harvested at different OD₆₀₀ values were subjected to ³¹P NMR analysis of changes in intracellular P_i (Fig. 1C) and polyphosphate (Fig. 1D) pools. In a composite of the results, it can be seen that the growth-dependent decrease in intracellular P_i of the wild-type and pho89 cells was highly similar, while the cellular P_i content of the pho84 cells was more than twofold lower at an OD₆₀₀ of 0.5. It is interesting that the twofold-lower content of intracellular free P_i coincided with an approximately twofold increase in extracellular P_i content. Moreover, the slight increase in intracellular free P_i observable when these cells had reached an OD₆₀₀ of 1.5 was paralleled by a decrease in extracellular P_i content (Fig. 1B). As in the case of the intracellular content of P_i, both wild-type and pho89 cells maintained polyphosphates at significant and comparable levels at an OD₆₀₀ of 0.5 while the polyphosphate content was close to exhausted at higher OD₆₀₀ values. In contrast, pho84 cells, which initially had a slightly lower polyphosphate content, had, at an OD₆₀₀ of 1.5, accumulated a high level of polyphosphates which, at an OD₆₀₀ of 3, had been reduced to a level comparable to that of wild-type and pho89 cells. The P_i acquisition by pho84 cells following the initial rapid efflux was during prolonged growth (OD₆₀₀ of 1.5) paralleled by a pronounced synthesis of intracellular polyphosphate known to occur under conditions where phosphate and metabolic energy are available, especially when P_i is added to cells previously starved for P_i, resulting in intracellular P_i levels of up to 20 µmol/g (wet weight) of cells (2, 13). It has been suggested that when, with continued growth, the metabolic requirements of the cells exceed the extracellular supply of P_i which can be taken up via the P_i transporters, vacuolar polyphosphate is mobilized to replenish the cytosolic phosphate pool (1, 6).

In summary, the results presented in this work reveal that regulation of the Pho84p P_i transport activity does not require the participation of the Pho89p, as the transport activity of the Pho84p in pho89 cells is regulated as in the wild-type cells. Interestingly, both pho84 cells and double-disruptant cells, devoid of a high-affinity transport system active at pH 4.5, catalyze an apparent rapid efflux of internal P_i. The obtained results suggest that the activation of the Pho84p transporter and that of the Pho89p transporter are independently regulated, with activation and inactivation of the Pho89p transport activity early in the growth phase and the Pho84p transport, in contrast, maximally active at mid-log phase.