Intracellular Phosphate Serves as a Signal for the Regulation of the PHO Pathway in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) is known to regulate the expression of several phosphate-responsive genes, such as PHO5 and PHO84. However, the fundamental issue of whether cells sense intracellular or extracellular phosphate remains unresolved. To address this issue, we have directly measured intracellular phosphate concentrations by 31 P NMR spectroscopy. We find that PHO5 expression is strongly correlated with the levels of both intracellular orthophosphate and intracellular polyphosphate and that the signaling defect in the ⌬pho84 strain is likely to result from insufficient intracellular phosphate caused by a defect in phosphate uptake. Furthermore, the ⌬phm1⌬phm2, ⌬phm3, and ⌬phm4 strains, which lack intracellular polyphosphate, have higher intracellular orthophosphate levels and lower expression of PHO5 than the wild-type strain. By contrast, the ⌬phm5 strain, which has lower intracellular orthophosphate and higher polyphosphate levels than the wild-type strain, shows repressed expression of PHO5, similar to the wildtype strain. These observations suggest that PHO5 expression is under the regulation of intracellular orthophosphate, although orthophosphate is not the sole signaling molecule. Moreover, the disruption of PHM3, PHM4, or of both PHM1 and PHM2 in the ⌬pho84 strain suppresses, although not completely, the PHO5 constitutive phenotype by increasing intracellular orthophosphate, suggesting that Pho84p affects phosphate signaling largely by functioning as a transporter.Inorganic phosphate is an essential nutrient needed in large amounts for nucleic acid and phospholipid biosynthesis, as well as for energy metabolism. It is therefore essential that organisms have appropriate regulatory mechanisms to respond and adapt rapidly to changes in phosphate availability (1). In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) regulates the expression of several phosphate-responsive genes that are involved in the scavenging and specific uptake of phosphate from extracellular sources, including PHO5, which encodes repressible acid phosphatase (rAPase), 1 and PHO84, which encodes a high affinity phosphate transporter that acts in response to phosphate levels (2-4). Under high phosphate conditions, the transcription factor Pho4p is phosphorylated by the cyclin-dependent kinase (CDK) complex Pho80p-Pho85p (5) and exported from the nucleus to the cytoplasm (6), thereby turning off expression of the PHO5 and PHO84 genes. When yeast cells are starved of phosphate, the CDK inhibitor Pho81p inactivates 8). Under these conditions, Pho4p is unphosphorylated and active (9), leading to the induction of PHO5 and PHO84 expression to scavenge phosphate from the environment (2-4, 10). Although many components and the molecular mechanism of the PHO pathway have been extensively studied, fundamental questions, such as whether cells sense intracellular or extracellular phosphate and what phosphate metabolites function as signals for regulating t...

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…Phosphate uptake was significantly reduced in the pho84 mutant even at high-orthophosphate concentrations, thus suggesting that constitutive expression of PHO5 in the pho84 mutant could be due to decreased internal phosphate concentration (Wykoff and O'Shea 2001); This assumption was further supported by direct measurement of internal phosphate concentration by 31 P NMR (Auesukaree et al 2004;Pinson et al 2004). This regulatory defect of PHO5 expression in the pho84 mutant could be compensated by overexpressing low-affinity phosphate transporters, and thus it is unlikely that Pho84 acts as a critical phosphate sensor in the phosphate regulatory pathway (Wykoff and O'Shea 2001).…”

“…However, in work based on mutant analysis and use of agonists such as glycerol-3-phosphate, Pho84 was found to be involved in phosphate signaling to the protein kinase A pathway (Popova et al 2010). Importantly, a good correlation was observed between internal phosphate concentration and PHO5 expression in several mutants (Auesukaree et al 2004), thus suggesting that there is an internal phosphate-concentration-sensing mechanism. How it is connected to Pho81 regulation of the Pho80-85 kinase via an IP7-dependent and/or -independent mechanism remains to be determined.…”

Regulation and Properties of PstSCAB, a High-Affinity, High-Velocity Phosphate Transport System ofSinorhizobium meliloti

“…The Pho phenotype resulting from the pstC1021 mutation is similar to the Pho regulon constitutive phenotype that results from pst mutations in E. coli (20,71,73) and from mutations in the PHO84 high-affinity P i transport system in Saccharomyces cerevisiae (76). While these data suggest that the transport systems play a role in sensing the P i concentration and the signal transduction pathway, more recent evidence suggests that it is the intracellular concentration of P i that is the major signal for regulation of the Pho pathway (3,32,76). We note that the pstC1021 mutation of S. meliloti 1021 results in a partial Pho constitutive phenotype in which PhoB transcription of genes such as pstS is constitutive (Table 3), whereas transcription of the phoA gene encoding alkaline phosphatase and the phoC genes is repressed upon addition of 2 mM P i (5).…”

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity

“…The magnitude of this signal corresponded to the absolute increase in signal that the same EcPpk1 construct produced in a wild-type cell. Because yeast cells are known to contain ,200 mM polyP (Auesukaree et al, 2004), we can estimate that polyP synthesized by EcPpk1 might reach up to ,40 mM. EcPpk1-expressing cells showed a growth defect as compared with the wild-type cells (Fig.…”