R E S E A R C H A R T I C L E

CharacterizationofthePho89phosphate transporter by functionalhyperexpression inSaccharomyces cerevisiaeRenata A. Zvyagilskaya1, Fredrik Lundh2, Dieter Samyn2, Johanna Pattison-Granberg2, Jean-MarieMouillon2, Yulia Popova3,4, Johan M. Thevelein3,4 & Bengt L. Persson2

1A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect, Moscow, Russia; 2School of Pure and Applied Natural Sciences,

Kalmar University, Kalmar, Sweden; 3Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven,

Arenberg, Leuven-Heverlee, Flanders, Belgium; and 4Department 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 82 Kalmar, Sweden. Tel.:

146 480 446276; fax: 146 480 446262;

e-mail: bengt.persson@hik.se

Present addresses: Johanna Pattison-

Granberg, Sweden Recycling AB,

Jarnvagsgatan 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: Andre Goffeau

Keywords

phosphate uptake system; Pho89; PHO

pathway; Saccharomyces cerevisiae .

Abstract

The Na1-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 pho87Dpho90D 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 Km 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 Na1 ions and

sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of

H1- and Na1-coupled phosphate transport systems in wild-type cells grown at

different pH values was quantified. The contribution of the Na1-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 SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

high-affinity transport system (PHO84, PHO89) and for the

secreted acid phosphatases (PHO5, PHO11, PHO12). The

corresponding proteins are synthesized in order to scavenge

phosphate from the surrounding environment (Lenburg &

O’Shea, 1996; Oshima, 1997). High-capacity uptake of free

phosphate from outside the cell under phosphate-limiting

conditions is mainly mediated by the Pho84 transporter

(Bun-ya et al., 1991). In the absence of the high-affinity

transporters, the low-affinity transporters, Pho87, Pho90

and Pho91, are needed for phosphate transport but deletion

of these does not result in any major phosphate uptake

defect (Wykoff & O’Shea, 2001). Of these, the Pho91 protein

was recently proposed to be a vacuolar phosphate transpor-

ter active in the regulation of intracellular phosphate home-

ostasis and polyphosphate levels (Hurlimann et al., 2007).

Of the high-affinity transporters, Pho84 has been well

characterized previously and has been shown to scavenge

phosphate from the surrounding medium in a symport

manner with protons at an acidic pH optimum of 4.5

(Bun-ya et al., 1991). Although much less information is

available about the Pho89 transporter, it has been proposed

to have an alkaline pH optimum of 9.5 and phosphate

uptake coupled with cations, preferably Na1 ions (Martinez

& Persson, 1998). Of several identified sodium-dependent

phosphate transporters from both higher and lower eukar-

yotes (Werner & Kinne, 2001; Virkki et al., 2007), Pho89

belongs to the PiT family (Saier, 2000) also comprising the

extensively characterized mammalian homologs PiT-1 and

PiT-2 (Werner & Kinne, 2001; Collins et al., 2004).

It is believed that the Pho89 transporter plays a minor

role in total phosphate acquisition. In previous studies, we

have shown that under low-phosphate growth conditions,

the Pho89 transporter displays a 100-fold lower phosphate

uptake activity as compared with the Pho84 transporter

(Pattison-Granberg & Persson, 2000). Recently, whole-gen-

ome studies have revealed that the PHO89 gene is not only

upregulated under phosphate limitation but also by Mg21

starvation, cell wall damage, Ca21 stress and alkalinization

(Garcia et al., 2004; Viladevall et al., 2004; Wiesenberger

et al., 2007). When switching to alkaline conditions, PHO89

shows a strong calcineurin pathway-dependent transcrip-

tional induction already after 5–10 min (Serrano et al.,

2002). The derepression is believed to be caused by an

increase in the cytoplasmic Ca21 concentration that occurs

under these stress conditions (Yoshimoto et al., 2002).

Under alkaline extracellular conditions, phosphate entry is

mainly in the form of bivalent phosphate ions, HPO42�,

resulting in cytosolic alkalinization. Further support for the

alkaline response being caused by a Ca21 burst under

alkaline growth conditions was obtained by the demonstra-

tion that expression of b-galactosidase behind the PHO89

promoter was strongly affected by the intracellular Ca21

concentration (Viladevall et al., 2004). Under alkaline con-

ditions, phosphate acquisition via the Pho89 transporter is

favored by the upregulation of the Na1-ATPase Ena1

(Serrano et al., 2002), resulting in a generation of a Na1

motive force across the plasma membrane.

In a recent study, it has been proposed that the presence of

the low-affinity phosphate transporters (PHO87, PHO90 and

PHO91) is needed for normal phosphate repression of the

PHO-regulated genes (Pinson et al., 2004). In contrast to

repression in a wild-type (WT) strain, in a pho87D pho90Dpho91D strain the PHO genes are induced under high-phos-

phate conditions (Auesukaree et al., 2003; Pinson et al., 2004).

This finding is further supported by the observation that under

high-phosphate conditions a quadruple deletion strain expres-

sing only the Pho84 or the Pho89 transporter is still able to take

up phosphate and maintain growth (Wykoff & O’Shea, 2001;

Auesukaree et al., 2003). Interestingly, Wykoff et al. (2007) show

that overexpression of the negative regulator of the low-affinity

transport system, Spl2, induces expression from the PHO84

promoter, resulting in similar protein levels as in a pho87Dpho90D pho91D strain. The pho84D pho87D pho90D pho91Dquadruple deletion strain exhibits a higher rate of phosphate

uptake compared with a pho84D strain under both high- and

low-phosphate growth conditions. This indicates enhanced

activity and/or hyperexpression of the Pho89 transporter, which

is in agreement with the increased PHO89 transcript levels seen

in this strain (Auesukaree et al., 2003). In previous studies,

phosphate transport via hyperexpressed Pho89 was measured at

acidic pH without any addition of sodium, indicating that

Pho89 is apparently able to mediate phosphate uptake without

Na1-coupling at acidic pH. Growth capacity, however, under

these conditions was strongly reduced.

The results in the present study highlight the role and

contribution of Pho89 in the total phosphate acquisition system

under different growth conditions. For the first time, Pho89

expression was followed over a broad pH range and it was

clearly demonstrated that the level was enhanced under alkaline

conditions. We have also made use of the pho84D pho87Dpho90D pho91D quadruple deletion strain (Auesukaree et al.,

2003) to characterize in detail the transport properties of Pho89

without any influences of other Pi transporters. Although the

Pho89 expression level in the pho84D pho87D pho90D pho91Dstrain was constantly high during growth at pH values between

4.5 and 8.0, the transport activity was strongly diminished

under acidic conditions, implying some type of mechanistic

restraint. We have investigated in detail the ion-coupling

requirement of Pho89-mediated phosphate uptake in a strain

where the activity was not influenced by other phosphate

transporters and demonstrated that Na1 was the preferred

cotransported cation. Furthermore, for the first time, we have

also precisely determined the kinetic parameters, Km and Vmax,

under conditions optimal for Pho89 transport activity and also

presented evidence that the Pho89 transporter, in contrast to

Pho84 and Pho87 (Giots et al., 2003), is not able to sense

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

686 R.A. Zvyagilskaya et al.

external phosphate for activation of the protein kinase A (PKA)

pathway.

Materials and methods

Growth media, growth conditions and yeaststrains

Yeast cells were routinely grown at 30 1C in either high-

phosphate (HPi) or low-phosphate (LPi) liquid media as

described elsewhere (Mouillon & Persson, 2005) or on

complex buffered agar-solidified LPi medium containing

0.54% Bacto peptone, 2% glucose, 2% agar, 0.05% MgSO4,

0.02% MgCl2, 0.04% (NH4)2SO4, 0.1% NaCl, standard

concentrations of vitamins and microelements and c.

250 mM Pi as traces from the reagents. To starve cells for

phosphate, they were precultured at 30 1C into the mid-

exponential phase to an A600 nm of 1.0–1.5 in YP medium

(1% yeast extract, 2% bacto peptone) with 2% glucose. The

mid-exponential-phase cells were then harvested and trans-

ferred to phosphate starvation medium (5.7 g L�1 of yeast

nitrogen base without phosphate), pH 8.0, with 4% glucose

and appropriate amino acids. Cells were starved for 3 days at

30 1C under continuous shaking and the starvation medium

was refreshed daily.

Culture media were autoclaved, adjusted to the desired

pH values with H2SO4 or KOH and supplemented with

50 mM Tris-HCl buffer. Cultures confluent in the same

50 mM Tris-HCl buffers were aseptically collected by cen-

trifugation and suspended to 10 A600 nm U mL�1. Aliquots

(150mL) of the suspension were spread onto plates

(1.5 A600 nm U per plate) and allowed to grow for 18–23 h.

The validity of this method was demonstrated previously

(Zvyagilskaya et al., 2001; Zvyagilskaya & Persson, 2003); an

empirically optimized growth period on the solidified

buffered media was used to attain maximal activity of both

high-affinity H1- and Na1-coupled phosphate transport

systems. Cell growth was monitored turbidometrically at

A600 nm. Saccharomyces cerevisiae strains used in this study

are listed in Table 1.

Phosphate transport measurements

Phosphate uptake was assayed in intact S. cerevisiae cells

grown under LPi conditions. Cells were washed with 25 mM

Tris-succinate buffer adjusted to the pH of the assay buffer

to be used subsequently and resuspended in the same buffer

supplemented with 3% glucose, alkali ions and an uncou-

pler as indicated. The uptake was initiated by addition

to a 30mL cell suspension (0.546 mg dry weight) of 1 mL

of [32P]orthophosphate (carrier-free, 0.18 Cimmol�1,

1 mCi = 37 MBq; GE Healthcare, UK). Phosphate uptake

was terminated by addition of 3 mL ice-cold Tris-succinate

dilution buffer. The cell suspensions were immediately

filtered, the Whatman GF/F filters (Whatman, UK) were

washed once with the same cold dilution buffers and the

radioactivity retained on the filters was determined by liquid

scintillation spectrometry. Lineweaver–Burke analysis of

phosphate uptake as a function of external phosphate

concentration in WT (SH8246) and pho84D pho87D pho90Dpho91D (SH6302) strains was carried out with cells grown

on solid LPi media at pH 4.5 and 8.0, respectively. Initial

velocities at phosphate concentrations ranging from 1.71 to

11 mM were determined over the first 60 s in Tris-succinate

buffer (pH 4.5) supplemented with 3% glucose for SH8246

and Tris-succinate buffer (pH 8) supplemented with 3%

glucose and 10 mM NaCl for SH6302. For phosphate uptake

into intact phosphate-starved WT (SH8246) and pho84Dpho87D pho90D pho91D (SH6302) strains, cells were har-

vested by centrifugation and the pellet was washed twice

with 25 mM MES buffer adjusted to the appropriate pH.

Then, 120 mL of freshly washed cells was resuspended in

1.5 mL of phosphate-starvation medium at pH 4.5 and 8.0.

Aliquots of 80 mL were incubated for 15 min at 30 1C under

continuous shaking, after which 20mL of the [32P]ortho-

phosphate stock solution with a final concentration of 1 mM

KH2PO4 was added. Transport was terminated after 5 min

by adding 5 mL of ice-cold water. Cells were rapidly filtered,

rinsed twice with ice-cold water and filters were subjected to

liquid scintillation spectrometry as described above.

Sampling, extraction and determination oftrehalase activity

Phosphate-starved glucose-repressed cells were rapidly

cooled on ice and harvested by centrifugation. The pellet

was washed twice with ice-cold 25 mM 2-(N-morpholino)

ethanesulfonic acid (MES) buffer adjusted to the appropri-

ate pH and resuspended in phosphate-starvation medium,

Table 1. Saccharomyces cerevisiae strains used in this study

Strain Genotype Sources or references

SH8246 MATa ade2 leu2-3, 112 ura3-1, 2 trp1-289 his3-532 can1 pho3-1 Ogawa & Oshima (1990)

SH8333 MATa ade2 leu2-3, 112 ura3-1, 2 trp1-289 his3-532 can1 pho3-1 pho84<HIS3 Auesukaree et al. (2003)

SH6302 MATa ade2 leu2-3, 112 ura3-1, 2 trp1-289 his3-532 can1 pho3-1 pho84<HIS3 pho87<

URA3 pho90<CgTRP1 pho91<CgLEU2

Auesukaree et al. (2003)

FEMS Yeast Res 8 (2008) 685–696 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

687Characterization of the Pho89 phosphate transporter

pH 8.0, in the presence of 4% glucose, and incubated at

30 1C with shaking. After 30 min of incubation, KH2PO4

was added to the culture. Samples (75 mg cells mL�1) were

withdrawn at indicated time points. Cells were rapidly

cooled by addition of ice-cold water, centrifuged and

resuspended in 500 mL of ice-cold 25 mM MES buffer, pH

7.0. Crude cell extracts were prepared as described pre-

viously (Pernambuco et al., 1996) and dialyzed (BRL micro-

dialysis system) against 25 mM MES buffer, pH 7, 50 mM

CaCl2 at 4 1C. Trehalase activity in dialyzed cell extracts was

determined using a coupled enzymatic reaction of glucose

oxidase and peroxidase with glucose as described previously

(Pernambuco et al., 1996).

RNA isolation and relative quantitative reversetranscriptase (RT)-PCR

Total RNA from yeast cells grown at pH 4.5 at 30 1C to an

A600 nm of 1.0 in either HPi (30 mM) or LPi (250mM) liquid

media (Kaneko et al., 1982) was isolated using RNAqueousTM

(Ambion). RNA samples were treated with RQ1 RNAse-free

DNAse (Promega) before the RT-PCR reaction using the

SuperScriptTM One-step RT-PCR system (Invitrogen, the

Netherlands). Primers (50-AATGCTTTACTGCTGCTTGG

and 50-AGGTCCACTTCCTGGATGTC) and (50-TGAGGT

TGCTGCTTTGGTA and 50-TTCTGGGGCTCTGAATCTTT)

were used for amplification of a 430-bp fragment of the

PHO89 gene and a 769-bp fragment of the actin (ACT1) gene,

respectively. One hundred nanograms of total RNA was used

for each reaction. The linear range of the PCR amplification in

terms of cycle numbers and substrates has been preliminarily

checked for both PHO89 and ACT1 transcripts. Amplified

DNA products were separated on a 1% agarose gel and

transcript levels were quantified based on the signal intensity

detected in the presence of ethidium bromide using the MULTI-

ANALYST software (BioRad).

Electrophoresis and Western blot analysis

Cells were collected from liquid and plate cultures. Proteins

were extracted and precipitated as described previously

(Horak & Wolf, 2001). Equivalent concentrations of the

resolubilized protein (30 mg) were mixed with sample buffer

before separation on a 10% sodium dodecyl sulfate-poly-

acrylamide gel (Laemmli et al., 1970). Immunoblotting onto

poly(vinylidene difluoride) membranes (Immobilon-P,

Millipore) was carried out according to the manufacturer’s

protocol. The Pho89 antibody was produced in rabbit

against a peptide corresponding to the consensus sequence

of 12 residues (YYEGRRNLGTTV) located in the large

cytosolic loop connecting TM7 and TM8, and purified

using affinity chromatography (Innovagen, Sweden). Horse-

radish peroxidase-conjugated anti-rabbit-IgG-antibody (GE

Healthcare) was used for immunological detection of the

Pho84 and Pho89 proteins. After a short incubation with the

chemiluminescent substrate, the blot was exposed to an

X-ray film for 1–2 min. The molecular masses of separated

proteins were determined by comparison with the relative

mobility of prestained marker proteins (Fermentas, Germany).

Phosphate determinations

Phosphate in the growth media was assayed spectrophoto-

metrically at 850 nm essentially as described (Nyren et al.,

1986).

Results

Pho89 protein expression level and activity arehighly elevated in a pho84D pho87D pho90Dpho91D strain in agreement with the elevatedtranscript level

The goal of these studies was to establish PHO89 hyper-

expressive growth conditions, allowing us to analyze in a

comprehensive manner the functional properties and reg-

ulation of the Pho89 transporter. To start, we performed an

RT-PCR to validate PHO89 expression levels in three

different strains, i.e. WT (SH8246), pho84D (SH8333) and

pho84D pho87D pho90D pho91D (SH6302), grown under

HPi and LPi conditions. As shown in Fig. 1, PHO89

transcripts were only barely detected in WT cells grown on

HPi or LPi media. In contrast, in the pho84D strain the

transcript level was significantly increased, especially under

LPi conditions where the level was approximately twofold as

compared with the HPi-grown cells. In the pho84D pho87Dpho90D pho91D strain, the transcript level further increased

strongly both in Lpi- and in HPi-grown cells. The loss of

phosphate dependence in transcriptional induction of

Fig. 1. Relative quantification of PHO89 transcripts by RT-PCR. Strains,

WT (SH8246), pho84D (SH8333) and pho84D pho87D pho90D pho91D(SH6302) grown in both HPi and LPi media were subjected to RT-PCR

performed on total RNA collected from cells using synthesized homo-

logous primers directed towards the PHO89 gene and the actin gene

(ACT1) as an internal standard.

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

688 R.A. Zvyagilskaya et al.

PHO89 in the strain carrying the quadruple deletion is in

agreement with a previous report (Auesukaree et al., 2003).

We next investigated whether the upregulated PHO89

transcript level in the quadruple deletion mutant strain also

conferred high protein expression and high-capacity phos-

phate transport in cells grown on solid buffered LPi medium

at pH 7.0. As shown in Fig. 2, phosphate-uptake activity

assayed at pH 8.5 in the presence of 5 mM NaCl increased

progressively and reached its maximum (c. 7 mmol min�1 g�1

cells) at an A600 nm of 22 of the culture, coinciding with

maximal protein expression of the Pho89 transporter.

Further cell growth resulted in a decrease of both protein

expression and activity of the Pho89 transporter. The

dramatic upregulation of functionally active Pho89 shown

in this study resulted in a c. 100-fold higher transport

activity than reported previously (Pattison-Granberg &

Persson, 2000).

Pho89 protein expression and activity is stronglypH dependent

Next, we have analyzed in detail the pH-dependence of

Pho89 activity and protein expression in the quadruple

deletion mutant (Fig. 3). Although cells of this strain grown

on solid LPi media at pH 4.5, 6.0 and 8.0 revealed efficient

synthesis and only a slightly increased expression of the

Pho89 protein in the pH range of 4.5–8.0, the phosphate

uptake activity of cells grown at pH 4.5, 6.0 and 8.0 was

Fig. 2. Time course of the evolution of [32P]orthophosphate uptake

activity by pho84D pho87D pho90D pho91D (SH6302) cells during their

growth on agar solidified buffered LPi medium, pH 7.0. Cells grown

overnight on solidified buffered LPi medium, pH 7.0, were aseptically

collected, washed twice with sterile 25-mM Tris-succinate buffer, pH 7.0,

resuspended in the same buffer and spread on solidified LPi medium, pH.

7.0 (’). At specified time points, cells were harvested by centrifugation

at 5500 g for 15 min and washed three times with phosphate- and

substrate-free medium adjusted to pH 8.5. Phosphate uptake (�) was

measured in 25-mM Tris-succinate buffer in the presence of 3% glucose

and 10-mM NaCl at pH 8.5. Average and SE values of triplicate assays

using the same cell preparation are plotted. Lower panel: immuno-

detection of the Pho89 protein levels in samples collected at the

indicated time points.

Fig. 3. Activity and protein levels of phosphate uptake as a function of

pH. pH dependence of phosphate uptake by pho84D pho87D pho90Dpho91D (SH6302) cells grown on agar solidified buffered LPi media. Cells

grown at pH 4.5, 6.0 and 8.0 for 18.5 h were collected from the plates,

washed and resuspended in cold Tris-succinate buffer at appropriate pH

values. Phosphate uptake into cells grown at pH 4.5 (’), 6.0 (.) and 8.0

(m) was measured at different pH values (pH 3.5–10) in 25-mM Tris-

succinate buffer in the presence of 3% glucose and 10-mM NaCl.

Average and SE values of triplicate assays using the same cell preparation

are plotted. Lower panel: immunodetection of Pho89 protein expressed

in cells grown at pH 4.5, 6.0 and 8.0 for 18.5 h.

FEMS Yeast Res 8 (2008) 685–696 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

689Characterization of the Pho89 phosphate transporter

highly pH-dependent, with a much lower phosphate trans-

port efficiency in cells grown at pH 4.5 as compared with

cells grown at pH 6.0 or 8.0. The results suggest that even if

the protein is synthesized and routed to the membrane over

this broad pH range, its functionality is strictly pH depen-

dent. Independent of whether cells were grown at pH 4.5, 6.0

or 8.0, the optimum pH of transport was 7.5 or higher,

consistent with the reported alkaline pH optimum of

Pho89-mediated phosphate transport in WT cells (Martinez

& Persson, 1998).

The Pho89 permease is a Na1-coupledphosphate transporter highly induced underalkaline conditions

Because Pho89 is the sole Pi uptake system in the quadruple

deletion mutant, we analyzed its dependence on alkali ions,

glucose and proton coupling at pH 8.5. For this purpose, we

used cells in which phosphate uptake was maximal, i.e. cells

grown at pH 8.0 on solid LPi medium to an A600 nm of 28

(Fig. 4a). In agreement with earlier observations on the

alkali-dependent Pi uptake in WT cells (Martinez & Persson,

1998), Na1 was found to be the preferred coupling ion with

a twofold advantage over K1 and Li1. The Na1-coupled

phosphate uptake activity of Pho89 in the quadruple dele-

tion mutant showed saturable uptake kinetics with Km and

Vmax values for Na1 coupling of 0.71� 0.01 mM and

2.2� 0.11mmol min�1 g�1 cells, respectively (calculated

from the double reciprocal plot, Fig. 5 and inset). Omission

of alkali ions or glucose (in the presence of Na1) reduced

Na1-coupled Pho89 activity six- and twofold, respectively

(Fig. 4a). Because the phosphate uptake activity, monitored

in the presence of Na1, was approximately threefold re-

duced in the presence of carbonyl cyanide m-chlorophenyl-

hydrazone (CCCP) (Fig. 4a), it appears that dissipation of

the proton gradient exerts a limitation on Na1 coupling of

the Pho89 transporter. In contrast, in WT cells grown at pH

6.0, phosphate uptake activity measured at pH 4.5 was

glucose dependent with no Na1 stimulation (Fig. 4b).

Phosphate uptake at pH 4.5 was reduced fivefold in the

presence of CCCP, stressing the tight proton coupling of the

phosphate uptake system under these conditions.

Determination of kinetic parameters (Km and Vmax) of

phosphate uptake by Pho84 and Pho89 in WT (Fig. 6a) and

quadruple mutant (Fig. 6b) cells grown on solid LPi media

at pH 4.5 and 8.0, respectively, was accomplished by

phosphate uptake measurement in the presence of glucose

at pH 4.5 (WT) and at pH 8.0 in the presence of the Na1

(quadruple deletion) strain. The calculated Km and Vmax

values for Pho84 at pH 4.5 and Pho89 at pH 8.0 were

24.64� 0.02mM and 5.56� 0.09 mmol min�1 g�1 cells and

37.68� 0.01mM and 7.32� 0.14 mmol min�1 g�1 cells,

respectively.

The distinct characteristics of phosphate transport in WT

cells grown at pH 4.5 and 8.0, i.e. at two extremes of the pH

growth range, prompted us to quantify the contribution of

the H1- and Na1-coupled phosphate uptake systems to

Fig. 4. (a) Effect of alkali ions, glucose and CCCP on phosphate uptake

by pho84D pho87D pho90D pho91D (SH6302) cells grown on agar

solidified buffered LPi media, pH 8.0. pho84D pho87D pho90D pho91D(SH6302) cells grown on LPi YPD plates at pH 8.0 for 16 h were collected

from the plates, washed and resuspended in cold Tris-succinate buffer at

pH 8.5. Phosphate uptake was measured in Tris-succinate buffer (pH 8.5)

supplemented with 3% glucose in the absence (� ) and presence of

5-mM NaCl (’), 5-mM KCl ( ), 5-mM LiCl (^), 5-mM NaCl and 60-mM

CCCP (�), and 5-mM NaCl without glucose ( ). Average and SE values of

triplicate assays using the same cell preparation are plotted. (b) Effect of

alkali ions, glucose and CCCP on phosphate uptake by WT (SH8246) cells

grown at pH 6.0 in LPi YPD medium. WT cells grown in LPi YPD medium

at pH 6.0 for 4.5 h were harvested, washed and resuspended in Tris-

succinate buffer, pH 4.5. Pi uptake was measured in 25-mM Tris-

succinate buffer at pH 4.5 in the absence of glucose (,), in the presence

of 3% glucose (m), in the presence of 3% glucose and 10-mM NaCl ( )

and in the presence of 3% glucose and 60-mM CCCP (�). Average and SE

values of triplicate assays using the same cell preparation are plotted.

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

690 R.A. Zvyagilskaya et al.

phosphate uptake in cells grown on buffered solid LPi media

at different pH values. Phosphate uptake was assayed at pH

4.5 (optimal for the Pho84 system) and 8.0 (optimal for the

Pho89 system) in the presence and absence of 5 mM NaCl.

Activity at pH 4.5 without Na1 was taken as a measure of

the Pho84 system, while the activity in the pH 8.0 buffer

supplemented with 5 mM NaCl was taken as a measure of

the Pho89 system. As shown in Fig. 7, the H1-coupled

transport provides most of the phosphate uptake over the

pH range of growth. The contribution of the Na1-coupled

transport system to the total cellular phosphate uptake

increases progressively with increasing pH, reaching its

maximum in cells grown at pH 8.0. However, H1-coupled

transport is also maintained at pH 8.0, presumably as a

consequence of the broad pH range of Pho84-mediated

Fig. 5. Stimulatory effect of NaCl on phosphate uptake by pho84Dpho87D pho90D pho91D (SH6302) cells grown on LPi YPD plates at pH

8.2. pho84D pho87D pho90D pho91D (SH6302) cells grown on LPi YPD

plates at pH 8.2 for 19 h were collected from the plates, washed and

resuspended in cold Tris-succinate buffer at pH 8.0. Phosphate uptake

was measured in 25-mM Tris-succinate buffer, pH 8.0, supplemented

with 3% glucose and various concentrations of NaCl (0–20 mM) (’).

Average and SE values of triplicate assays using the same cell preparation

are plotted. The inset shows a double-reciprocal plot of the same data

points.

Fig. 6. Lineweaver–Burk plots describing Pi uptake by Pho84 and Pho89

in WT (a) and pho84D pho87D pho90D pho91D (b) cells as a function of

external Pi concentrations. (a) WT cells grown on solid LPi YPD plates at

pH 4.5 were collected, washed and resuspended in cold Tris-succinate

buffer at pH 4.5. Phosphate uptake was measured in 25-mM Tris-

succinate buffer, pH 4.5, supplemented with 3% glucose and various

concentrations of Pi. (b) pho84D pho87D pho90D pho91D cells grown

on solid LPi YPD plates at pH 8.0 were collected, washed and resus-

pended in cold Tris-succinate buffer at pH 8.0. Phosphate uptake was

measured in 25 mM Tris-succinate buffer, pH 8.0, supplemented with

3% glucose, 5-mM NaCl and various concentrations of Pi. Initial

velocities at Pi concentrations varying from 1.71 to 11 mM were deter-

mined over the first 60 s. Each point represents the mean of three

determinations using the same cell preparation.

FEMS Yeast Res 8 (2008) 685–696 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

691Characterization of the Pho89 phosphate transporter

uptake. A comparison of the pH dependence on the expres-

sion of the Pho89 transporter in WT cells grown at a

rigorously maintained pH of 4.5, 6.0, 7.0 or 8.0 under LPi

conditions also revealed a clear pH dependence with a

preferred pH of 7.0 and higher (Fig. 7, lower panel).

Notably, in contrast to the situation in the quadruple

deletion mutant, expression of Pho89 in the WT cells was

clearly suppressed at pH 4.5.

Phosphate activation of the PKA pathway

Because Pho89 is able to take over the role of the main

phosphate supplier in the quadruple deletion strain, we next

wished to examine whether Pho89 could also act as a

phosphate sensor in this mutant strain. It has recently been

shown that both Pho84 and Pho87 can act as phosphate

sensors for activation of the PKA pathway when phosphate

is added to cells starved previously for phosphate (Giots

et al., 2003). Pho84-mediated phosphate sensing and signal-

ing for activation of the PKA pathway seems to trigger the

removal of Pho84 from the plasma membrane in response to

elevated external phosphate concentrations (Mouillon &

Persson, 2005). Activation of the PKA pathway was mea-

sured by assaying trehalase activity as one of its principal

targets as described previously (Giots et al., 2003). Following

phosphate starvation, a high concentration of phosphate

(10 mM) was added to WT or the quadruple deletion

mutant cells. As can be seen in Fig. 8, no activation of

trehalase was detected in cells carrying the quadruple dele-

tion. This implies that even under conditions of hyperex-

pression where Pho89 has taken over the role of Pho84 as a

high-capacity uptake system, Pho89 can apparently not act

as a sensor for the activation of the PKA signaling pathway

unlike the Pho84 protein (Giots et al., 2003; Mouillon &

Persson, 2005),

Discussion

In S. cerevisiae, the ability to take up Pi from a phosphate-

limited environment is largely dependent on activities of the

high-affinity phosphate transporters, Pho84 and Pho89,

with Pho84 serving as the main high-capacity uptake system

in the normal habitat of this yeast. Although this transporter

has an acidic pH optimum, it is also highly active in the

neutral and alkaline pH ranges, partially overlapping with

the activity of the Pho89 transporter. Typically, in a WT

strain of S. cerevisiae, the maximal activity of the Pho89

transporter is about 100-fold lower than that of the Pho84

transporter (Pattison-Granberg & Persson, 2000). The

contribution of Pho89 to the total phosphate uptake is

rather limited even at alkaline pH values. For this reason,

the precise role of this transporter has remained obscure

and also its regulation and kinetics have been less well

characterized.

While PHO89 transcript levels are very low in a WT strain

under normal growth conditions, both a single deletion

strain (pho84D) and a quadruple deletion strain (pho84Dpho87D pho90D pho91D) exhibit an elevated PHO89 tran-

script level under both high- and low-Pi conditions (Fig. 1)

in agreement with other studies (Auesukaree et al., 2003).

Based on this, the quadruple deletion (pho84D pho87Dpho90D pho91D) strain was considered as a suitable model

strain for investigating the properties of the Pho89 trans-

porter in greater detail. First we showed that the higher

PHO89 transcript levels are associated with higher protein

and activity levels of Pho89. This allowed, for the first time,

a detailed characterization of the Pho89 transporter and its

effect on phosphate-regulated pathways. It is noteworthy

Fig. 7. Contribution of H1- and Na1-coupled phosphate transport

systems to the total phosphate uptake by WT cells (SH8246) grown on

LPi YPD plates at pH values ranging from 4.5 to 8.0. A600 nm of WT cells

was determined by suspending cells grown on two plates in 10 mL of the

appropriate buffer. Cells grown to an A600 nm of c. 36 on solid LPi media

at pH 4.5, 6.0, 7.0 and 8.0, respectively, were collected from the plates,

washed and resuspended in cold Tris-succinate buffer at pH 8.0.

Phosphate uptake was measured in 25-mM Tris-succinate buffer supple-

mented with 3% glucose, pH 4.5 (open bars), and in 25-mM Tris-

succinate buffer, pH 8.0, supplemented with 3% glucose and 15-mM

NaCl (filled bars), respectively. Average and SE values of triplicate assays

using the same cell preparation are plotted. Lower panel: immuno-

detection of Pho89 protein expressed in cells grown for 18.5 h on solid

LPi media at pH 4.5, 6.0, 7.0 and 8.5.

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

692 R.A. Zvyagilskaya et al.

that the deletion of the low-affinity transporters, in line with

previous observations (Pinson et al., 2004), releases the

expression of PHO89 from the PHO pathway control,

resulting in elevated PHO89 transcription also under nor-

mally repressive HPi conditions. In agreement with the

transcription data of Fig. 1, the Pho89 protein levels were

transiently highly induced in a growth-dependent manner

(Fig. 2, lower panel). Under optimal conditions, maximal

protein expression was accompanied by a maximal phos-

phate uptake rate in the range of 7 mmol min�1 g�1 cells, the

highest value reported so far for S. cerevisiae. This can be

explained by the high level of Pho89 expressed in this strain

and also possibly, in part, by a trapped content of inorganic

phosphate due to an increased activity of the secreted acid

phosphatase Pho5, shown previously to be derepressed in

the quadruple deletion (pho84D pho87D pho90D pho91D)

(Auesukaree et al., 2003; Mouillon & Persson, 2005) and in

the pho84D strain (Pattison-Granberg & Persson, 2000).

Although the Pho89 transporter has been shown pre-

viously to have an alkaline pH optimum and a highly

inducible transcript level during growth at alkaline pH

values, a correlation with high protein levels under these

conditions has not been shown. We have shown (Fig. 3,

lower panel) that although the Pho89 protein is stably

expressed in the quadruple deletion mutant over a wide pH

range, with a slightly higher protein expression level under

alkaline conditions, Pho89-mediated transport activity (Fig.

3, upper panel) does not correlate with the protein expres-

sion levels, being restricted only to the neutral/alkaline pH

range. The effect of pH on phosphate transport can have

several causes. The permease could be selective for bivalent

phosphate ions as shown for sodium-coupled transport in

mammals (Moschen et al., 2001). Another possibility might

be that protons and Na1 bind to the same transmembrane

domain with a possible conformational change as has been

proposed for mammalian type II Na1/Pi transporter (de la

Horra et al., 2000) or an effect on the charge distribution

within the transporter itself. Although Pho89-mediated

phosphate uptake is difficult to estimate in the WT strain,

we have, using an elaborated method allowing cells to grow

at rigorously maintained pH values, clearly demonstrated

that the protein is expressed and highly induced under

neutral and alkaline growth conditions in this strain (Fig.

7). This is in contrast to the situation in the quadruple

deletion strain where a similar expression level of Pho89 is

seen over a broad pH range of 4.5–8.0 (Fig. 2). At increased

pH values, the contribution of the Na1-stimulated Pho89

transport to the total uptake of phosphate is elevated (Fig.

7). This demonstrates the overlap between the H1-coupled

Pho84 and Na1-coupled Pho89 high-affinity uptake sys-

tems, which are both active under phosphate-limiting con-

ditions. Together, these findings suggest that Pho89

transports phosphate using a highly selective symport

mechanism and that Pho89 has the design and functional

properties to make it an important phosphate transporter in

this yeast in spite of the organism’s natural preference for an

acidic/neutral habitat. We investigated the properties and

regulation of the phosphate transport mediated by Pho89 in

detail.

By studying Pi uptake in the quadruple deletion mutant,

we have been able to establish its strong Na1-dependence in

comparison with other cations, e.g., Li1 and K1 (Fig. 4a)

and we have compared this with its behavior observed in the

WTstrain (Fig. 4b). The presence of glucose was required for

the high transport activity both in WT and in quadruple

mutant cells, independent of the preference of the two

transport systems. The strong effect of glucose is not likely

Fig. 8. Phosphate-induced activation of trehalase and Pi uptake in WT

and in pho84D pho87D pho90D pho91D cells. (a) Pi was added to

phosphate-starved cells at pH 4.5 or pH 8.0 and trehalase activity was

measured as a function of time. The specific activity of WTcells at pH 4.5

( ) and pH 8.0 (n) and of pho84D pho87D pho90D pho91D cells at pH

4.5 (m) and pH 8.0 (�) is expressed as nanomoles of glucose released per

minute and milligram of protein. (b) Pi uptake activity measured under

the same conditions in WTcells at pH 4.5 (light gray bar) and pH 8.0 (dark

gray bar) and in pho84D pho87D pho90D pho91D at pH 4.5 (black bar)

and pH 8.0 (white bar).

FEMS Yeast Res 8 (2008) 685–696 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

693Characterization of the Pho89 phosphate transporter

to be mediated by transcriptional regulation. It is known

that glucose positively regulates plasma membrane H1-

ATPase by inducing conformational changes (Lecchi et al.,

2005). H1-ATPase creates the proton electrochemical gra-

dient across the plasma membrane that is required for ionic

homeostasis and the uptake of nutrients (Serrano, 1991).

Disturbance of ionic homeostasis by reduced activity of the

H1-ATPase in the absence of glucose can result in reduction

of phosphate uptake. The protonophore CCCP had an effect

in both strains. The reason for the pronounced decrease in

Pi uptake into the quadruple deletion strain at pH 8.5 may

be due to either a direct effect on a possible H1-coupling of

the Pho89 transporter, an indirect effect mediated via other

H1-coupled membrane transporter or a combination here-

of. Besides its role in collapsing the proton electrochemical

gradient, CCCP was recently found to influence the phos-

pholipid distribution between the inner and the outer layers

of the plasma membrane (Stevens & Nichols, 2007).

Although inhibition of the flip–flop of phospholipids might

inhibit Pi uptake mediated by the H1-coupled as well as the

Na1-coupled transport system, it is less likely that this

process is fast enough to influence the uptake under the

conditions used in this study.

The kinetic parameters of the Pho89 transporter under

optimal hyperexpressive conditions were evaluated (Fig. 5).

When assayed for phosphate uptake at pH 8.0 the Km and

Vmax values for Pho89 expressed in the quadruple deletion

were 37.68� 0.01 mM and 7.32� 0.14 mmol min�1 g�1 cells,

values close to those of Pho84 in WT cells (24.64� 0.02 ıM

and 5.56� 0.09 mmol min�1 g�1 cells, respectively) when

phosphate uptake was assayed at for Pho84 optimal condi-

tions (Fig. 6a and b). The Km value for the Na1-coupled

uptake system in WT cells was reported previously to be

0.6–1 mM at pH 7.2 (Roomans et al., 1977; Roomans &

Borst-Pauwels, 1979). The Km and Vmax values for

the Pho89 Na1-coupling were 0.71� 0.01 mM and

2.24� 0.11mmol min�1 g�1 cells, respectively (Fig. 5).

We also investigated the correlation between phosphate

transport and signaling in the phosphate-starved cells ex-

pressing Pho89 at different pH values. Measurement of Pi

transport has shown that at alkaline pH, the quadruple

deletion mutant has a strong phosphate uptake while at

acidic pH it displayed c. 10-fold lower transport activity

(Fig. 8). This result again indicated a strong pH dependence

of Pho89 activity. On the other hand, phosphate-induced

trehalase activation was abolished in the quadruple deletion

mutant under both conditions. This result indicates that

despite improved transport activity at alkaline pH, Pho89

cannot provide efficient signaling of phosphate availability

for activation of the PKA pathway. Moreover, these data

clearly serve as evidence that transport of phosphate is not

enough for mediation of rapid phosphate signaling. It also

demonstrates that phosphate is not sensed intracellularly in

the cells expressing only Pho89. Uptake in WT cells was

almost not affected by differences in the pH, reflecting a

broad adaptation capability of yeast cells in this regard.

Phosphate-induced trehalase activation was also found to be

independent of pH. Most probably, phosphate signaling in

this case was mediated by the Pho84 transporter (Giots

et al., 2003), which is active at a broad pH range. The

absence of signaling with Pho89 further supports the con-

cept that rapid phosphate signaling for activation of the

PKA pathway in yeast is mediated by specific phosphate

transporters–sensors or transceptors, like Pho84 and Pho87

(Giots et al., 2003). This situation is similar to rapid amino

acid signaling for activation of the PKA pathway where

especially the Gap1 amino acid carrier appears to mediate

the process as a transceptor (Donaton et al., 2003).

Acknowledgements

This work was supported by grants from the Swedish

Research Council (621-2003-3558) and the Faculty Fund of

Kalmar University to B.L.P.; by Russian Foundation for

Basic Research (grant 06-04049687) and Russian Academy

of Sciences (cellular and molecular biology) grants to

R.A.Z.; grants from the Fund for Scientific Research –

Flanders and the Research Fund of the Katholieke Universi-

teit Leuven (Concerted Research Actions) to J.M.T.; a travel

fellowship to DS from Petra and Karl-Erik Hedborgs Foun-

dation; and a Marie Curie fellowship to Y.P. from E.C. F.L.

was supported by a PhD program within the Graduate

Research School in Pharmaceutical Sciences at Kalmar

University. We thank Prof. Satoshi Harashima (University

of Osaka, Japan) for providing the yeast strains SH8246,

SH8333 and SH6302 used in this study.

References

Almaguer C, Mantella D, Perez E & Patton-Vogt J (2003) Inositol

and phosphate regulate GIT1 transcription and

glycerophosphoinositol incorporation in Saccharomyces

cerevisiae. Eukaryot Cell 2: 729–736.

Auesukaree C, Homma T, Kaneko Y & Harashima S (2003)

Transcriptional regulation of phosphate-responsive genes in

low-affinity phosphate-transporter-defective mutants in

Saccharomyces cerevisiae. Biochem Biophys Res Commun 306:

843–850.

Bun-ya M, Nishimura M, Harashima S & Oshima Y (1991) The

PHO84 gene of Saccharomyces cerevisiae encodes an inorganic

phosphate transporter. Mol Cell Biol 11: 3229–3238.

Collins JF, Bai L & Ghishan FK (2004) The SLC20 family of

proteins: dual functions as sodium-phosphate cotransporters

and viral receptors. Pflugers Arch 447: 647–652.

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

694 R.A. Zvyagilskaya et al.

de la Horra C, Hernando N, Lambert G, Forster I, Biber J &

Murer H (2000) Molecular determinants of pH sensitivity of

the type IIa Na/P(i) cotransporter. J Biol Chem 275:

6284–6287.

Donaton MC, Holsbeeks I, Lagatie O, Van Zeebroeck G, Crauwels

M, Winderickx J & Thevelein JM (2003) The Gap1 general

amino acid permease acts as an amino acid sensor for

activation of protein kinase A targets in the yeast

Saccharomyces cerevisiae. Mol Microbiol 50: 911–929.

Garcia R, Bermejo C, Grau C et al. (2004) The global

transcriptional response to transient cell wall damage in

Saccharomyces cerevisiae and its regulation by the cell integrity

signaling pathway. J Biol Chem 279: 15183–15195.

Giots F, Donaton MC & Thevelein JM (2003) Inorganic

phosphate is sensed by specific phosphate carriers and acts in

concert with glucose as a nutrient signal for activation of the

protein kinase A pathway in the yeast Saccharomyces cerevisiae.

Mol Microbiol 47: 1163–1181.

Horak J & Wolf DH (2001) Glucose-induced

monoubiquitination of the Saccharomyces cerevisiae galactose

transporter is sufficient to signal its internalization. J Bacteriol

183: 3083–3088.

Hurlimann HC, Stadler-Waibel M, Werner TP & Freimoser FM

(2007) Pho91 is a vacuolar phosphate transporter that

regulates phosphate and polyphosphate metabolism in

Saccharomyces cerevisiae. Mol Biol Cell 18: 4438–4445.

Kaneko Y, Toh-e A & Oshima Y (1982) Identification of the

genetic locus for the structural gene and a new regulatory gene

for the synthesis of repressible alkaline phosphatase in

Saccharomyces cerevisiae. Mol Cell Biol 2: 127–137.

Laemmli UK, Beguin F & Gujer-Kellenberger G (1970) A factor

preventing the major head protein of bacteriophage T4 from

random aggregation. J Mol Biol 47: 69–85.

Lagerstedt JO, Voss JC, Wieslander A & Persson BL (2004)

Structural modeling of dual-affinity purified Pho84 phosphate

transporter. FEBS Lett 578: 262–268.

Lecchi S, Allen KE, Pardo JP, Mason AB & Slayman CW (2005)

Conformational changes of yeast plasma membrane H(1)-

ATPase during activation by glucose: role of threonine-912 in

the carboxy-terminal tail. Biochemistry 44: 16624–16632.

Lenburg ME & O’Shea EK (1996) Signaling phosphate starvation.

Trends Biochem Sci 21: 383–387.

Martinez P & Persson BL (1998) Identification, cloning and

characterization of a derepressible Na1-coupled phosphate

transporter in Saccharomyces cerevisiae. Mol Gen Genet 258:

628–638.

Moschen I, Setiawan I, Broer S, Murer H & Lang F (2001) Effect

of NaPi-mediated phosphate transport on intracellular pH.

Pflugers Arch 441: 802–806.

Mouillon JM & Persson BL (2005) Inhibition of the protein

kinase A alters the degradation of the high-affinity phosphate

transporter Pho84 in Saccharomyces cerevisiae. Curr Genet 48:

226–234.

Nyren P, Nore BF & Baltscheffsky M (1986) Studies on

photosynthetic inorganic pyrophosphate formation in

Rhodospirillum rubrum chromatophores. Biochim Biophys Acta

851: 276–282.

Ogawa N & Oshima Y (1990) Functional domains of a positive

regulatory protein, PHO4, for transcriptional control of the

phosphatase regulon in Saccharomyces cerevisiae. Mol Cell Biol

10: 2224–2236.

Oshima Y (1997) The phosphatase system in Saccharomyces

cerevisiae. Genes Genet Syst 72: 323–334.

Pattison-Granberg J & Persson BL (2000) Regulation of cation-

coupled high-affinity phosphate uptake in the yeast

Saccharomyces cerevisiae. J Bacteriol 182: 5017–5019.

Pernambuco MB, Winderickx J, Crauwels M, Griffioen G, Mager

WH & Thevelein JM (1996) Glucose-triggered signalling in

Saccharomyces cerevisiae: different requirements for sugar

phosphorylation between cells grown on glucose and those

grown on non-fermentable carbon sources. Microbiology 142:

1775–1782.

Persson BL, Lagerstedt JO, Pratt JR, Pattison-Granberg J, Lundh

K, Shokrollahzadeh S & Lundh F (2003) Regulation of

phosphate acquisition in Saccharomyces cerevisiae. Curr Genet

43: 225–244.

Pinson B, Merle M, Franconi JM & Daignan-Fornier B (2004)

Low affinity orthophosphate carriers regulate PHO gene

expression independently of internal orthophosphate

concentration in Saccharomyces cerevisiae. J Biol Chem 279:

35273–35280.

Roomans GM & Borst-Pauwels GWFH (1979) Interaction of

cations with phosphate-uptake by Saccharomyces cerevisiae –

effects of surface potential. Biochem J 178: 521–527.

Roomans GM, Blasco F & Borst-Pauwels GW (1977) Cotransport

of phosphate and sodium by yeast. Biochim Biophys Acta 467:

65–71.

Saier MH Jr (2000) A functional-phylogenetic classification

system for transmembrane solute transporters. Microbiol Mol

Biol Rev 64: 354–411.

Serrano R (1991) Transport across yeast vacuolar and plasma

membranes. The Molecular and Cellular Biology of the Yeast

Saccharomyces: Genome Dynamics, Protein Synthesis, and

Energetics (Broach JR, Jones EW & Pringle JR, eds), pp.

523–585. Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, NY.

Serrano R, Ruiz A, Bernal D, Chambers JR & Arino J (2002) The

transcriptional response to alkaline pH in Saccharomyces

cerevisiae: evidence for calcium-mediated signalling. Mol

Microbiol 46: 1319–1333.

Stevens HC & Nichols JW (2007) The proton electrochemical

gradient across the plasma membrane of yeast is necessary for

phospholipid flip. J Biol Chem 282: 17563–17567.

Viladevall L, Serrano R, Ruiz A, Domenech G, Giraldo J, Barcelo

A & Arino J (2004) Characterization of the calcium-mediated

response to alkaline stress in Saccharomyces cerevisiae. J Biol

Chem 279: 43614–43624.

Virkki LV, Biber J, Murer H & Forster IC (2007) Phosphate

transporters: a tale of two solute carrier families. Am J Physiol

Renal Physiol 293: 643–654.

FEMS Yeast Res 8 (2008) 685–696 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

695Characterization of the Pho89 phosphate transporter

Werner A & Kinne RK (2001) Evolution of the Na-P(i)

cotransport systems. Am J Physiol Regul Integr Comp Physiol

280: 301–312.

Wiesenberger G, Steinleitner K, Malli R, Graier WF, Vormann J,

Schweyen RJ & Stadler JA (2007) Mg21 deprivation elicits

rapid Ca21 uptake and activates Ca21/calcineurin signaling in

Saccharomyces cerevisiae. Eukaryot Cell 6: 592–599.

Wykoff DD & O’Shea EK (2001) Phosphate transport and sensing

in Saccharomyces cerevisiae. Genetics 159: 1491–1499.

Wykoff DD, Rizvi AH, Raser JM, Margolin B & O’Shea EK

(2007) Positive feedback regulates switching of

phosphate transporters in S. cerevisiae. Mol Cell 27:

1005–1013.

Yoshimoto H, Saltsman K, Gasch AP et al. (2002) Genome-wide

analysis of gene expression regulated by the calcineurin/Crz1p

signaling pathway in Saccharomyces cerevisiae. J Biol Chem

277: 31079–31088.

Zvyagilskaya R & Persson BL (2003) Dual regulation of proton-

and sodium-coupled phosphate transport systems in the

Yarrowia lipolytica yeast by extracellular phosphate and pH.

IUBMB Life 55: 151–154.

Zvyagilskaya R, Parchomenko O, Abramova N, Allard P,

Panaretakis T, Pattison-Granberg J & Persson BL (2001)

Proton- and sodium-coupled phosphate transport systems

and energy status of Yarrowia lipolytica cells grown in acidic

and alkaline conditions. J Membr Biol 183: 39–50.

FEMS Yeast Res 8 (2008) 685–696c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

696 R.A. Zvyagilskaya et al.