Phosphate Transport and Sensing in Saccharomyces cerevisiae

Corresponding author: Erin K. O'Shea, Howard Hughes Medical Institute, Department of Biochemisty and Biophysics, University of California, 513 Parnassus Ave., San Francisco, CA 94143-0448., oshea{at}biochem.ucsf.edu (E-mail)

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84 strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84 cells; however, the pho84 strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84 cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84 strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.

INORGANIC phosphate is an essential nutrient required for the synthesis of nucleic acids, phospholipids, and cellular metabolites. Reactions that synthesize these compounds require millimolar concentrations of phosphate whereas most environmental concentrations are substantially lower. To concentrate phosphate in the cytoplasm, cells utilize phosphate transporters. During phosphate starvation the capacity for phosphate uptake increases (PERSSON et al. 1999 ). In Saccharomyces cerevisiae this increase in phosphate uptake, as well as many other phosphate starvation responses, is regulated by the phosphate signal transduction pathway (PHO pathway; LENBURG and O'SHEA 1996 ). Many components of the PHO pathway are well understood; however, little is known about how external phosphate concentrations are sensed or what role phosphate uptake has in phosphate sensing.

The PHO pathway (reviewed in LENBURG and O'SHEA 1996 ; OSHIMA 1997 ) in budding yeast has been genetically defined by assaying for the activity of Pho5p, a broad specificity acid phosphatase, whose transcription is induced in response to phosphate starvation. Central to the PHO pathway is a cyclin/cyclin-dependent kinase complex (CDK; Pho80p/Pho85p) whose activity is regulated in response to external phosphate concentrations (KAFFMAN et al. 1994 ). Pho81p, a CDK inhibitor, binds to the cyclin-CDK when cells are grown in both high and low phosphate conditions but only markedly inhibits the kinase in vivo during phosphate starvation (SCHNEIDER et al. 1994 ; OGAWA et al. 1995 ). Mutations that inactivate the cyclin-CDK lead to constitutive expression of PHO5, and null mutations in the CDK inhibitor result in the inability to induce PHO5 (LEMIRE et al. 1985 ). Cells defective in Pho84p, a phosphate starvation-inducible high-affinity H⁺/PO₄ symporter, also constitutively express PHO5; genetically, PHO84 is upstream of all other PHO pathway components, suggesting a role for phosphate transport in phosphate sensing (BUN-YA et al. 1991 ).

Pho84p shares 39% similarity with both Snf3p and Rgt2p, which are 12 transmembrane domain-containing proteins in yeast involved in signaling information about external glucose concentrations (OZCAN et al. 1996 ; COSTANZO et al. 2001 ). Snf3p and Rgt2p share significant identity with hexose transporters; however, key experiments have demonstrated that Snf3p and Rgt2p do not participate in glucose transport but instead sense glucose, leading to the proper regulation of at least seven hexose transporters (THEODORIS et al. 1994 ; OZCAN et al. 1996 ; COONS et al. 1997 ; KRUCKEBERG et al. 1998 ; OZCAN and JOHNSTON 1999 ). Overexpression of hexose transporters in a snf3 strain allows for growth on low glucose-containing media but does not suppress the snf3 regulatory defect, indicating Snf3p has a role distinct from glucose uptake (COONS et al. 1997 ; OZCAN and JOHNSTON 1999 ). Furthermore, expression of SNF3 is insufficient to allow for growth on glucose when seven genes encoding hexose transporters (HXT1–7) are inactivated, suggesting that Snf3p does not directly participate in hexose uptake (LIANG and GABER 1996 ; OZCAN et al. 1998 ). An example of a transporter that acts as a sensor is the PstA protein in Escherichia coli (WANNER 1993 ). PstA is part of the phosphate transporter complex that regulates a two-component regulatory system composed of PhoR, a histidine protein kinase, and PhoB, a transcription factor. Certain mutations in the Pst transport complex, specifically in PstA, lead to defects in phosphate uptake without any disruption in phosphate starvation signaling through PhoR (COX et al. 1988 ).

We began these studies to determine whether Pho84p plays a role in phosphate sensing and to understand the role of phosphate uptake in signaling phosphate starvation. Our data indicate that Pho84p is an important phosphate transporter for growth under both high and low phosphate conditions and that Pho84p is not essential for sensing external phosphate concentrations. We also characterized phosphate transport in yeast cells and identified a synthetic lethal interaction between five phosphate transporters.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and growth conditions:
For strains with gene disruptions, all genes but PHO84 and PHO3 were inactivated using a PCR-based inactivation protocol that deleted the entire open reading frame (KITADA et al. 1995 ). PHO84 was inactivated using plasmid pMB123 (BUN-YA et al. 1991 ). PHO3 inactivation was described previously (HASWELL and O'SHEA 1999 ). Strain EY917 was generated by growing EY916 to stationary phase, plating 10⁷ cells on 5-fluoroorotic acid-containing media, and checking individual colonies for a stable Ura^- phenotype. Inactivation of the appropriate genes was confirmed by PCR and in some cases by Southern analysis. To generate combinations of mutations, standard genetic crossing, sporulation, and dissection techniques were used (GUTHRIE and FINK 1991 ). Yeast strains used in this study are listed in Table 1. All strains were grown in synthetic media with either 2% glucose (SD) or 2% galactose (SG) and appropriate amino acids. Media containing no phosphate were prepared as described except buffering agent was omitted (LAU et al. 2000 ). Strains EY916 and EY917 accumulated suppressors at a frequency of 10^-6. Most strains were maintained in SG-trp media with plasmid EB1280 (the GAL1 promoter driving PHO84 expression) to prevent the accumulation of suppressors. Strains without the five phosphate transporters were considered synthetic lethal because spores lacking all five transporters were unable to germinate on glucose-containing media. EY916 and EY917 cells are able to form very small colonies on glucose-containing media after 7 days at 30°.

Plasmids:
Plasmid EB1247 was constructed using a plasmid clone containing the full-length PHO87 cDNA (LIU et al. 1992 ). The cDNA was excised with BamHI and SacI and ligated to a HindIII and SacI fragment of pRS426 (CHRISTIANSON et al. 1992 ) and a 740-bp HindIII-BamHI-digested PCR product upstream (from 0 to 730 bp) of the ADH1 gene. EB1280 was constructed by digesting EB819 with BamHI and EcoRI to recover PHO84 and then ligating this with pRS314 (cut with KpnI and EcoRI) and the GAL1 promoter released with KpnI and BamHI from pRS316-GAL1-cDNA (SIKORSKI and HIETER 1989 ; LIU et al. 1992 ; LAU et al. 2000 ). Plasmids EB1341 (PHO89), EB1342 (PHO90), EB1368 (PHO84), EB1372 (PHO91), and EB1374 (GIT1) were constructed identically, using a 5' BamHI PCR primer and a 3' NotI PCR primer with gene-specific sequences that included the entire open reading frame (ORF). PCR products were digested and ligated with pRS426 that contained the same ADH1 promoter as EB1247. Because EB1341 did not completely suppress all transport defects (discussed later), PHO89 was sequenced from this plasmid and there were no mutations.

Phosphate uptake:
Strains were grown to log phase (OD₆₀₀ = 0.5–1.0) in SG-trp media, transferred to SD-trp media, and grown for 3 hr. Cells were rapidly washed (<3 min) three times with SD media containing no phosphate, resuspended in 0.9 ml of no phosphate media with 4% glucose, and incubated at 30° for 1 min prior to addition of a 0.1-ml KH₂³²PO₄ (Perkin Elmer Life Sciences, Boston) mixture at pH 4.5. The amount of phosphate uptake by cells was measured for 5–10 min (during which time the uptake was linear) by filtering the cells from the media with a 0.45-µm nitrocellulose filter (Millipore, Bedford, MA) and washing the cells with 3 ml of 0.5 M KH₂PO₄. Uptake was calculated by measuring the amount of radioactivity in the cells at t = 1 min and t = 6 min by scintillation counting, converting uptake of counts per minute into nanomoles phosphate, and normalizing to cell density. The specific activity of the phosphate mixture ranged from 1 x 10⁵ to 3 x 10⁵ cpm nmol^-1 phosphate. Six different concentrations of phosphate (5 mM, 1 mM, 0.5 mM, 0.1 mM, 50 µM, and 10 µM) were used to derive an aggregate V_max and K_m from reciprocal plots (SEGEL 1975 ). The R² values for linear regression of the reciprocal plots exceeded 0.90 except for strain EY916, which had R² values >0.50. The K_m and V_max were determined using Lineweaver-Burk plots, which are less susceptible to skewing as a consequence of multiple kinetics components, i.e., high- and low-affinity uptake; therefore, both the K_m and V_max are aggregate values reflecting the contribution of many individual kinetic constants.

Fluorescence microscopy:
PHO84-GFP was constructed using homologous recombination of a PCR product (LONGTINE et al. 1998 ). Proper integration was confirmed by colony PCR and by direct visualization of green fluorescent protein (GFP). This strain behaved like wild type when assayed for phosphatase activity (LAU et al. 1998 ). A BX60 microscope was used with a CCD camera (Photometrics, Tucson, AZ) to capture live-cell images (Olympus, Lake Success, NY). Images were processed minimally with Adobe Photoshop 5.0.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The pho84 strain is defective in phosphate uptake in high phosphate conditions:
Two models for Pho84p function can explain the constitutive expression of phosphate-responsive genes observed in a pho84 strain. In the first model, Pho84p senses high phosphate concentrations and sends a signal through the PHO pathway to repress PHO5 expression. The second model suggests that pho84 cells may be defective in phosphate uptake even in high phosphate conditions, resulting in a state of continued phosphate starvation. To differentiate between these two models, we measured the rate of phosphate uptake in cells grown in high phosphate. Previous studies in budding yeast have examined steady-state phosphate uptake in cells grown under phosphate limiting conditions (BUN-YA et al. 1991 ; LAU et al. 1998 ; OGAWA et al. 2000 ). We grew cells in standard synthetic high phosphate (7.1 mM) media, rapidly washed external phosphate from the cells, replaced the media with a known amount of phosphate and a ³²PO₄ tracer, and measured the amount of phosphate uptake in a short time course. This approach allowed for an approximation of the initial velocity of phosphate uptake as a function of exogenous phosphate concentrations and provided an estimate for the K_m and V_max of phosphate uptake for a given strain (Fig 1). One OD₆₀₀ unit of wild-type yeast cells (1 x 10⁷ cells) transports on average 2.5 nmol phosphate min^-1 in high phosphate media, whereas the rate of phosphate uptake in pho84 cells is one-half as much. Thus, pho84 cells may constitutively express phosphate starvation genes because they are starved for phosphate.

View larger version (24K): In this window In a new window Download PPT slide   Figure 1. (A) Uptake of phosphate as a function of phosphate concentration. Strains EY57 (wild type) and EY105 (pho84), both containing EB1280 (pGAL1-PHO84), were grown to midlog phase in SG-trp media, transferred to SD-trp media, and grown for 3 hr. After washing the cells of external phosphate, the amount of phosphate uptake by cells was measured and converted into an uptake velocity, the units of which are nmol phosphate min-1 OD600-1. The diamonds and squares represent the means of phosphate uptake for wild-type and pho84 cells, respectively, at the given phosphate concentrations for three experiments. The error bars are the standard deviations. (B) Reciprocal plot of phosphate uptake data. The diamonds and squares represent the reciprocals of the means of wild-type and pho84 cells, respectively.

Overexpression of phosphate transporters suppresses the signaling and phosphate uptake defects of a pho84 strain:
Because pho84 cells are defective in uptake in high phosphate conditions, we hypothesized that by increasing the amount of intracellular phosphate through overexpression of phosphate transporters we would suppress the constitutive PHO5 phenotype. Previously, overexpression of vascular plant phosphate transporters has been shown to suppress both the uptake and signaling defect of pho84 cells (MUCHHAL et al. 1996 ). However, these experiments were carried out with transporters that share amino acid similarity with PHO84. Thus, it is possible that Pho84p is involved in signaling phosphate starvation and that the plant transporters suppress the signaling defect because they are similar enough to carry out the Pho84p signaling function. We chose to overexpress PHO87, PHO89, and two genes that have >30% identity with PHO87, PHO90 (YJL198w) and PHO91 (YNR013c), using a highly expressed constitutive promoter (ADH1). These four proteins do not share significant amino acid similarity or identity with Pho84p (<16% identity; THOMPSON et al. 1994 ).

Whereas Pho84p has been shown to be a high-affinity H⁺/PO₄ symporter, the role of Pho87p, Pho89p, Pho90p, and Pho91p in phosphate transport is less well defined (BERHE et al. 1995 ; FRISTEDT et al. 1999 ). Pho89p was identified as an ortholog of a Neurospora crassa Na⁺/PO₄ high-affinity symporter, and subsequently, experiments have confirmed that Pho89p functions in phosphate transport (MARTINEZ and PERSSON 1998 ); however, relative to Pho84p, Pho89p was shown to play a relatively minor role in phosphate uptake under standard conditions (PATTISON-GRANBERG and PERSSON 2000 ). PHO87 was identified in an arsenate resistance selection (BUN-YA et al. 1996 ). Because arsenate resistance is often associated with decreased phosphate uptake and Pho87p has significant similarity to human Na⁺/PO₄ symporters, Pho87p was hypothesized to transport phosphate into yeast cells.

Overexpression of PHO84, PHO87, PHO89, PHO90, or PHO91 increased the amount of phosphate uptake in pho84 cells when grown in high phosphate, but PHO89 overexpression resulted in the smallest increase (Table 2). The inefficient uptake conferred by PHO89 overexpression is likely a consequence of the conditions under which the uptake assays were performed. Uptake measurements are conducted in synthetic media (pH 4.5 without exogenous sodium), and experiments have demonstrated that Pho89p optimally functions at pH 9 with 25 mM Na⁺ (MARTINEZ and PERSSON 1998 ). When we performed phosphate uptake measurements with this strain at pH 9, Pho89p was functional with a K_m of 3 µM, but the cells grew poorly under these conditions (data not shown). Interestingly, only overexpression of PHO84 results in a restoration of the K_m to wild-type levels in synthetic media, suggesting that the other transporters have a lower affinity for phosphate than Pho84p under our experimental conditions.

View this table: In this window In a new window   Table 2. Suppression of uptake defect in pho84 strain when phosphate transporters are overexpressed

To determine if increased intracellular phosphate results in suppression of the constitutive PHO5 phenotype, we measured acid phosphatase activity in each of the strains with overexpressed phosphate transporters (BUN-YA et al. 1991 ). There was a strong correlation between strains that had increased uptake of phosphate (Table 2) and suppression of the defect in acid phosphatase expression (Fig 2). Furthermore, these strains were able to induce PHO5 expression in response to phosphate starvation (data not shown). These data indicate that appropriate repression of the PHO pathway in high phosphate conditions can occur independent of PHO84. Importantly, PHO87, PHO90, and PHO91 have no significant sequence similarity to PHO84, suggesting that Pho84p does not contain a domain critical for signaling. These observations suggest that the defect in gene expression in the pho84 strain results from a defect in phosphate uptake. Thus, it seems unlikely that Pho84p is the phosphate sensor that regulates the PHO pathway.

View larger version (29K): In this window In a new window Download PPT slide   Figure 2. Acid phosphatase activity of pho84 strains with overexpressed phosphate transporters. Wild type is EY915, and the pho84 strain is EY914. Empty vector is pRS426 (CHRISTIANSON et al. 1992 ), and the plasmids containing transporters are described in MATERIALS AND METHODS. All strains contained pGAL1-PHO84 (EB1280) and were grown in SG-trp-ura, transferred to SD media for 10 hr to shut off expression of PHO84, and allowed to grow to midlog phase prior to assaying for acid phosphatase activity. Hydrolysis of -nitrophenolphosphate was monitored by OD420 and normalized to cell density OD600 (BUN-YA et al. 1991 ). Three independent cultures were grown and the values were averaged. The error bars indicate standard deviations.

Five phosphate transporters are responsible for growth of yeast in high phosphate media:
To assess the role of each transporter in phosphate uptake in cells grown in high phosphate conditions, we inactivated PHO84, PHO87, PHO89, PHO90, and PHO91. pho84 or pho87 pho89 pho90 pho91 strains grew almost as well as a wild-type strain in standard synthetic media; however, when all five genes were inactivated we observed synthetic lethality (Fig 3). This inviability can be suppressed by growing cells in galactose-containing media in the presence of a plasmid containing PHO84 under the control of the GAL1 promoter. Furthermore, constitutive overexpression of PHO84, PHO87, PHO89, PHO90, or PHO91 resulted in vigorous growth (Fig 3), confirming that the synthetic-lethal relationship was dependent on these five transporters.

View larger version (35K): In this window In a new window Download PPT slide   Figure 3. Synthetic lethality of the pho84 pho87 pho89 pho90 pho91 strain. Row one is wild type (EY57), row two is the quintuple delete strain (EY917), and rows three through eight are EY917 containing various overexpression constructs. All strains also contain EB1280 (pGAL1-PHO84). Strains with vector contain pRS426 and the overexpression constructs are described in MATERIALS AND METHODS. Strains were grown to an OD600 between 0.3 and 0.6 in SG complete media, diluted to OD600 = 0.3, and plated in threefold dilutions. Both plates were incubated at 30° for 2 days.

To investigate whether the synthetic lethality is a consequence of the loss of phosphate transport, we overexpressed GIT1, an organic phosphate transporter. Git1p has been demonstrated to transport glycerophosphoinositol (GroPIns) into the cell (PATTON-VOGT and HENRY 1998 ); we expected that we could restore viability by growing these cells in GroPIns-containing media. Interestingly, overexpression of GIT1 restored viability in the synthetic-lethal strain independent of GroPIns but only in the presence of high phosphate (Fig 3). When we overexpressed Git1p in the pho84 strain both the uptake defect and PHO5 constitutive phenotype were suppressed (Fig 2 and Table 2). Suppression of the pho84 phenotypes and restoration of viability to the synthetically lethal strain by GIT1 overexpression indicate that Git1p can function as an inorganic phosphate transporter and that the quintuple delete strain is inviable because it is defective in phosphate uptake.

Contribution of each phosphate transporter to growth:
To understand the contribution of each transporter to total phosphate uptake in high phosphate-grown cells, we generated a quintuple transporter deletion strain kept alive by PHO84 under the control of an inducible promoter and examined the growth and phosphate uptake of strains that expressed one wild-type transporter under the control of its native promoter. Strains with only wild-type PHO87, PHO89, or PHO90 grew significantly slower than wild type but still supported growth on high phosphate media (Fig 4). The strain expressing only PHO91 grew very slowly but eventually formed colonies on solid media. While PHO91 allows for minimal growth under its native promoter, it allows for vigorous growth when overexpressed under the ADH1 promoter; a similar phenomenon is observed with overexpression of some hexose transporters (REIFENBERGER et al. 1997 ). We conclude from these data that under standard high phosphate conditions PHO84 makes the most important contribution to phosphate uptake; PHO87, PHO89, and PHO90 play less important roles in phosphate uptake; and that PHO91 plays a minimal role in phosphate uptake.

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. Growth of transporter mutants. Strains were grown and plated as described in Fig 3. Both plates were incubated at 30° for 2 days. The strains are described in Table 1 and Table 3. EY57, wild type, is ade2-1, while the other strains are ADE2, explaining the darker pigment on the glucose plate; the ADE2 genotype did not affect the results (data not shown). All strains contain pGAL1-PHO84 (EB1280).

To quantify the contributions of each transporter to phosphate uptake, we measured the kinetics of phosphate uptake in these quadruple delete strains (Table 3). Except for PHO84, single inactivation of any transporter had minimal effects on the V_max of phosphate uptake (data not shown). Inactivation of multiple transporters resulted in defects in phosphate uptake that correlated with growth phenotypes (compare Fig 4 with Table 3). The V_max of phosphate uptake in the PHO89 strain was considerably lower than expected based on the growth of this strain on plates, but this discrepancy is likely because phosphate uptake assays are performed at pH 4.5 for 5 min and growth on plates is assayed over a 2-day period.

Pho84p compensates for a loss of other phosphate transporters:
All of the strains that were inactivated for PHO84 expressed PHO5 constitutively. We attempted to generate a PHO5 constitutive phenotype independent of PHO84 by inactivating phosphate transporters other than PHO84. This quadruple transporter deletion strain containing wild-type PHO84 did not constitutively express acid phosphatase activity (EY918, Table 3). Furthermore, this strain grew well (Fig 4), had a high V_max of phosphate uptake, and had a very low K_m relative to wild-type cells (Table 3). This strain appeared to compensate for the lack of non-Pho84p-mediated phosphate uptake through the induction of Pho84p activity. To determine if this kinetic change was a consequence of Pho84p accumulation at the plasma membrane, we examined the localization of a Pho84-GFP fusion protein (LAU et al. 2000 ). In wild-type cells, little fluorescence from Pho84-GFP is observed in high phosphate-grown cells, but Pho84-GFP accumulates at the plasma membrane and vacuole in cells starved for phosphate. Remarkably, the pho87 pho89 pho90 pho91 cells with Pho84-GFP are intensely fluorescent; Pho84-GFP is localized to the plasma membrane and the vacuole (Fig 5). The vacuolar fluorescence is likely a consequence of Pho84-GFP endocytosis (LAU et al. 2000 ). These data in combination with the kinetic data suggest that increases in Pho84p activity can compensate for a loss of other phosphate transporters. We examined PHO84 transcript levels with northern analysis and observed only a 1.2- to 2-fold increase in the amount of steady-state transcript (data not shown), suggesting that the accumulation of Pho84p at the plasma membrane involves post-transcriptional mechanisms. The mechanism of this compensation is unclear and warrants further study.

View larger version (34K): In this window In a new window Download PPT slide   Figure 5. Fluorescence microscopy of PHO84-GFP. Strains EY923 and EY924 were grown to OD600 = 0.6, washed three times with no phosphate media, resuspended in high or no phosphate media at an OD600 = 0.1, and grown for 4 hr prior to microscopy. Photomicrographs were captured under the same exposure conditions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have identified an important role for Pho84p in the uptake of phosphate in cells grown under high phosphate conditions. Previous work has shown that Pho84p is essential during phosphate starvation (BUN-YA et al. 1991 ); however, we demonstrate a role for Pho84p in phosphate uptake under high phosphate conditions and provide a transport-based explanation for the signaling defect of pho84 cells. Cells that are defective in PHO84 are unable to uptake enough phosphate to repress PHO5 expression; essentially, pho84 cells are always starving for phosphate. This idea is supported by the uptake data of pho84 cells grown in high phosphate and by the suppression of the signaling defect by unrelated phosphate transporters. It seems unlikely that both Pho87p and Pho89p, which share no significant amino acid similarity with Pho84p or with each other, contain common signaling domains capable of regulating the PHO pathway. Furthermore, overexpression of PHO89 leads to only a modest increase in phosphate uptake and only partially suppresses the signaling defect of pho84 cells. We conclude that Pho84p does not have a direct role in sensing external phosphate.

What factors are responsible for sensing phosphate concentrations and regulating the Pho80/Pho85 complex? Our studies suggest that the phosphate sensor is intracellular. The suppression of the PHO5 constitutive phenotype in pho84 cells was correlated with increased intracellular phosphate, suggesting an internal phosphate sensor. Furthermore, it seems unlikely that the phosphate sensor is associated with the vacuole, the location of >30% of cellular phosphate in the form of polyphosphate, because mutants defective in the accumulation or degradation of polyphosphate have no PHO pathway signaling defects (OGAWA et al. 2000 ). It is possible that the CDK inhibitor, Pho81p, senses phosphate directly and regulates the CDK activity; however, recent experiments have demonstrated that an 80-amino-acid fragment of Pho81p is necessary and sufficient for binding Pho80p/Pho85p and inhibiting the complex in vivo when grown in low phosphate (HUANG et al. 2001 ). It seems unlikely that this small fragment also binds phosphate (or a phospho-compound) with enough specificity to regulate the PHO pathway. More experiments are required to determine the identity of the protein that senses phosphate and regulates the Pho80p-Pho85p-Pho81p complex.

In addition to defining the role of PHO84 in phosphate signaling, we have established a synthetic-lethal relationship between five phosphate transporters. Interestingly, overexpression of GIT1, previously identified as a GroPIns transporter, suppresses the inviability of the quintuple delete strain, which raises the question of how many transporters are capable of transporting phosphate into the cell. Approximately 25 predicted proteins in the yeast genome share at least 20% identity with Pho84p, and it is possible that other transporters thought to transport another compound may also transport phosphate (COSTANZO et al. 2001 ). Although PHO84 and GIT1 share sequence similarity, GIT1 has a significantly higher K_m for phosphate than PHO84 (Table 2). On the basis of DNA microarray expression analysis, it is worth noting that GIT1 transcript accumulates during phosphate starvation or conditions that mimic phosphate starvation (OGAWA et al. 2000 ; CARROLL et al. 2001 ). Our studies, along with the work of others, indicate that Git1p serves as both an organic and inorganic phosphate transporter (PATTON-VOGT and HENRY 1998 ); this broad specificity may help the cells to better scavenge phosphate when it is limiting. Isolating multicopy suppressors of the inviable strain may be valuable in determining what proteins are capable of transporting phosphate into the cell and in identifying phosphate transporters from other species.

We inactivated all of the phosphate transporters but PHO84 to determine if we could phenocopy constitutive PHO5 expression independent of PHO84 by lowering the amount of phosphate brought into the cell; however, there was not a significant increase in the amount of Pho5p activity in this quadruple delete strain. When the kinetics of phosphate uptake and PHO84-GFP were observed in this quadruple deletion strain, it became clear that Pho84p was stabilized at the plasma membrane relative to a wild-type strain. Northern analysis demonstrated only a subtle increase in the amount of PHO84 transcript, indicating that post-transcriptional mechanisms have a role in regulating Pho84p activity. This is not surprising given that Pho84p abundance is regulated by endocytosis and this regulation is independent of the PHO pathway (LAU et al. 2000 ). It is likely that the inactivation of the other phosphate transporters results in a partial phosphate starvation phenotype, but Pho84p is able to compensate for this loss of phosphate uptake and the cells do not induce most phosphate starvation-responsive genes.

	ACKNOWLEDGMENTS

We thank Doug Jeffery for isolation of the PHO87 cDNA and the initial observation that PHO87 overexpression suppresses constitutive activation of the PHO pathway. Special thanks to Adam Carroll, Jonathan Raser, Archana Belle, Rusty Howson, Muyule Liku, David Steger, and Meghan Byrne for careful reading of the manuscript and constructive comments. Further thanks to all current and past members of the O'Shea laboratory for encouragement and advice. This work was supported by a postdoctoral research fellowship GM20762 from the National Institutes of Health (D.D.W.), grant GM51377 from the National Institutes of Health (E.K.O.), and the Howard Hughes Medical Institute.

Manuscript received July 26, 2001; Accepted for publication September 18, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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