RESEARCH ARTICLE Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae Renata A. Zvyagilskaya 1 , Fredrik Lundh 2 , Dieter Samyn 2 , Johanna Pattison-Granberg 2 , Jean-Marie Mouillon 2 , Yulia Popova 3,4 , Johan M. Thevelein 3,4 & Bengt L. Persson 2 1 A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect, Moscow, Russia; 2 School of Pure and Applied Natural Sciences, Kalmar University, Kalmar, Sweden; 3 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Arenberg, Leuven-Heverlee, Flanders, Belgium; and 4 Department of Molecular Biology, VIB, Kasteelpark, Arenberg, Leuven-Heverlee, Flanders, Belgium Correspondence: Bengt L. Persson, School of Pure and Applied Natural Sciences, Kalmar University, S-391 82Kalmar, Sweden. Tel.: 146 480 446276; fax: 146 480 446262; e-mail: bengt.persson@hik.se Present addresses: Johanna Pattison- Granberg, Sweden Recycling AB, J¨ arnv ¨ agsgatan 19, S-360 51 Hovmantorp, Sweden. Jean-Marie Mouillon, Fluxome Sciences A/S, Diplomvej 378, D-2800 Kgs. Lyngby, Denmark. Received 19 December 2007; revised 5 May 2008; accepted 27 May 2008. First published online July 2008. DOI:10.1111/j.1567-1364.2008.00408.x Editor: Andr ´ e Goffeau Keywords phosphate uptake system; Pho89; PHO pathway; Saccharomyces cerevisiae . Abstract The Na 1 -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 pho84D pho87D pho90D pho91D 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 1 ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H 1 - and Na 1 -coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na 1 -coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH. Introduction In order to adapt to a fluctuating nutrient environment, unicellular organisms need to have specialized nutrient sensing and uptake mechanisms. In the yeast Saccharomyces cerevisiae, inorganic phosphate (Pi) acquisition is accom- plished by both low-affinity and high-affinity Pi transport systems (Persson et al., 2003). Also, the glycerophosphoino- sitol transporter (Git) has been shown to be able to act as a low-affinity inorganic phosphate transporter (Wykoff & O’Shea, 2001; Almaguer et al., 2003; Pinson et al., 2004). The low-affinity Pi transport system, composed of the Pho87, Pho90 and Pho91 permeases, has been shown to be independent of external phosphate concentration at the transcriptional level (Auesukaree et al., 2003). However, in a recent study, it was proposed that the low-affinity system is downregulated by the PHO pathway as a consequence of an upregulation of a negative regulator of this system, Spl2, under low-phosphate conditions (Wykoff et al., 2007). In contrast, the high-affinity phosphate transport system com- posed of Pho84, a proton-coupled phosphate transporter (Bun-ya et al., 1991; Lagerstedt et al., 2004), and Pho89, a cation-coupled phosphate transporter (Martinez & Persson, 1998; Pattison-Granberg & Persson, 2000), is upregulated by the PHO regulatory pathway in response to low external phosphate (Oshima, 1997; Persson et al., 2003). When cells encounter phosphate limitation in the external medium, the PHO pathway induces expression of the genes coding for the FEMS Yeast Res 8 (2008) 685–696 c  2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved