Characterization of the Pho89 phosphate transporter by functional hyperexpression inSaccharomyces cerevisiae

Abstract

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.

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

“…Both PHO84 and PHO89 were among the most highly inducible genes upon phosphate limitation (Ogawa et al 2000). While Pho84 cotransports phosphate with H + , Pho89 is a phosphate/Na + symporter and works most efficiently under basic conditions, which are not usual physiological conditions for yeast cells (Zvyagilskaya et al 2008). Indeed, on the basis of knockout experiments, Pho84 appears to be the major high-affinity phosphate transporter (Pattison-Granberg and Persson 2000).…”

Phosphate homeostasis in the yeast Saccharomyces cerevisiae, the key role of the SPX domain‐containing proteins

“…Under Pi sufficient condition, Pho84 is phosphorylated and ubiquitinated, resulting in its internalization and vacuolar degradation [48]. Pho89 has recently been demonstrated to be a sodium coupled Pi transporter, being mainly active at alkaline pH [49].…”

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…In S. cerevisiae, Pho84 is thought to mediate phosphate uptake with a pH optimum of 4.5, while Pho89 is proposed to have an optimum of pH 9.5 (18)(19)(20). We therefore analyzed the growth of all of the C. neoformans mutants at pHs 4.0, 5.4, and 9.0.…”

“…The high-affinity phosphate uptake system in S. cerevisiae consists of Pho84, which mediates proton-coupled cotransport, and Pho89, which performs sodium-coupled transport upon phosphate limitation (18)(19)(20). The transcription factors Pho4 and Pho2 regulate the expression of the PHO genes in response to activation by the cyclin-dependent kinase Pho85, the cyclin Pho80, and the cyclin-dependent kinase inhibitor Pho81 (with similar components in N. crassa) (15,(21)(22)(23)(24)(25)(26).…”