The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter.

M Bun-Ya, M Nishimura, S Harashima and Y Oshima Mol. Cell. Biol. 1991, 11(6):3229. DOI: 10.1128/MCB.11.6.3229.

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

Updated information and services can be found at: http://mcb.asm.org/content/11/6/3229 These include:
CONTENT ALERTS

Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

MOLECULAR AND CELLULAR BIOLOGY, June 1991, p. 3229-3238

Vol. 11, No. 6

0270-7306/91/063229-10$02.00/0 Copyright 1991, American Society for Microbiology

The PH084 Gene of Saccharomyces cerevisiae Encodes an Inorganic Phosphate Transporter

MASANORI BUN-YA, MAMORU NISHIMURA, SATOSHI HARASHIMA, AND YASUJI OSHIMA* Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan Received 4 January 1991/Accepted 26 March 1991
Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

The PH084 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PH084 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PH084 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PH084 transcription was regulated by P1 like those of the PHOS, PHO8, and PHO81 genes. A PH084-lacZ fusion gene produced j}-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PH084 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PH084 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

There are at least two systems for uptake of Pi in Saccharomyces cerevisiae, one with a low Km value (8.2 ,uM) for external Pi and the other with a high Km value (770 ,uM) (36). The low-Km system is repressed by Pi through the same system as for regulation of repressible acid phosphatase (rAPase; EC 3.1.3.2) (50) and requires the PH084 gene product (36, 41). The pho84 mutant cells produce rAPase even in medium containing a sufficiently high concentration of Pi to repress rAPase synthesis in the wild-type cells. An assay of Pi uptake with 32p; shows that the pho84 mutant cells have severely reduced activity (41). These observations suggest alternative possibilities for the function of PH084, namely, that it encodes a Pi transporter or a positive regulatory factor specific for expression of a gene encoding the Pi transporter. This communication reports evidence supporting the former idea, that PH084 encodes a Pi transporter. First, the nucleotide sequence of the PH084 gene indicates that it encodes a protein homologous to the glucose transporter and various other sugar transporters. Second, a PHO84-,B-galactosidase fusion protein is recovered in a membrane fraction of S. cerevisiae cells.
MATERIALS AND METHODS

Organisms and plasmids. The S. cerevisiae strains used are listed in Table 1. All of the strains were heterothallic, as they had the ho genotype. All except MB137, NBW5, MT8-1, and DKD-5D had the pho3-1 genotype to eliminate another acid phosphatase activity synthesized in high-Pi medium (48). Escherichia coli JA221 (7) and MV1184 (43) were used for manipulation of DNA. A gene library of S. cerevisiae, YCp50 "CEN BANK" A, constructed by partial digestion of genomic DNA of S. cerevisiae with Sau3AI and its ligation with YCp50 (30) at the unique BamHI site, was obtained from the American Type Culture Collection (Rockville, Md.). Plasmids pUC118 and pUC119 (43) were used for preparation of single-stranded DNAs for DNA sequencing
*

Corresponding author.
3229

with a helper phage, M13 K07 (43). Plasmid YCp50 and pTIl5 (46), the latter of which is derived from YCp50 connected with the EcoRI-SstI-KpnI-SmaI-BamHI-XbaISalI part of the multiple cloning site of pUC18 (47) and both of which are marked with the URA3 gene of S. cerevisiae, were used as low-copy-number vectors, and YIp5 (28) was used as an integrative vector. Plasmid YIpl (28) was used for preparation of a 1.7-kbp BamHI fragment of the S. cerevisiae HIS3 gene. Plasmid pSH530, a YEp-type plasmid bearing the HIS5-lacZ fused gene (24), was used as a control in studies on expression of a PH084-lacZ fused gene. The HIS5 DNA fragment of the fusion gene contains a 0.6-kbp 5' noncoding sequence and a 121-bp region encoding the N-terminal region of histidinol-phosphate aminotransferase (EC 2.6.1.9). A 1.0-kbp HindIIl fragment of the S. cerevisiae ACT] gene was prepared from plasmid pYA301 (10), obtained from D. Gallwitz, and used as a hybridization probe. The structures and constructions of the other plasmids used in this study are shown in Fig. 1. Media and genetic and analytical methods. The nutrient medium (YPAD; i.e., nutrient high-Pi medium), minimal high-Pi and low-Pi media, and synthetic glucose (SD) medium for S. cerevisiae and the LB and M9 media for E. coli were described previously (49). The nutrient low-Pi medium was prepared as described previously (15). For detection of rAPase activity of pho84 mutants on plates by a staining method based on the diazo-coupling reaction (38), YPAGly and SGly media were prepared by replacing 20 g of glucose per liter of YPAD and SD media, respectively, with 30 g of glycerol. Results of the determination of the phenotype of the constitutive synthesis of rAPase by staining colonies of a pho84 mutant on YPAD plates are usually ambiguous, but this phenotype could be clearly determined by staining colonies on YPAGly plates, for some unknown reason. Cells of S. cerevisiae were cultivated at 30C, and those of E. coli were cultivated at 37C. Standard methods (34) were used for genetic manipulations of S. cerevisiae. S. cerevisiae was transformed by the lithium-acetate method (14), and E. coli was transformed by the method of Morrison (21). An S. cerevisiae DNA fragment was integrated into an S. cerevi-

3230

BUN-YA ET AL.
TABLE 1. S. cerevisiae strains used

MOL. CELL. BIOL.

Strain

Genotypea

Source

P-28-24C AX49-4B NS216 NS219 MB137 MB159 MB191

MB192b
NBD2-1c NBD4-1 NBD80-ld NBD81-6De NBW5 NBW78
YAT965 MT8-1 DKD-5D
b

MATa pho3-1 MATot pho3-1 his4 MATa pho3-1 leu2-3,112 trpl ura3-52 pho84-1 MATa pho3-1 leu2-3,112 ura3-52 pho84-1 MATa leu2 pho84-1 MATa pho3-1 (PHO84 URA3) pho84-1 leu2-3,112 trpl ura3-52 MATa pho3-1 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 can] MATa pho3-1 Apho84::HIS3 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 cani MATa pho3-1 Apho2::HIS3 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 can] MATa pho3-1 Apho4::HIS3 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 cani MATa pho3-1 Apho8O::HIS3 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 cani MATa pho3-1 Apho8J::HIS3 ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 can] MATat ade2 leu2-3,112 his3-532 trpl-289 ura3-1,2 can] MATa/MATot pho3-J/pho3-1 ade2lade2 leu2-3,11211eu2-3,112 his3-5321his3-532 trpl-2891trpl-289 ura3-1,21ura3-1,2 canllcani MATa pho3-1 pho85-1 Ieu2 his4 MATa ade his3 leu2 trpl ura3 gal80::LEU2 MATa his3 leu2-3,112 trpl

Our stock (38) Our stock (15) Our stock Our stock Our stock This study This study This study Our stock Our stock (25) Our stock Our stock Our stock (25) Our stock

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

Our stock (49, 50) T. Fukasawa Our stock (12)

a The genetic symbols used are as described by Mortimer et al. (22). Strain MB192, a pho84 null mutant, is a haploid segregant from the diploid strain MB183 with an integrated 5.4-kbp BamHI-EcoRI fragment of pMB123 (Fig. 1). ' Strain NBD2-1, a pho2 null mutant, was constructed from strain NBW7 by gene replacement of the 1.2-kbp EcoRI segment of the PHO2 (i.e., GRFO) gene ORF (49) by the 1.4-kbp BamHI-XhoI HIS3 DNA fragment of S. cerevisiae derived from YIpl. d Strain NBD80-1, a pho8O null mutant, was constructed from strain NBW7 by gene replacement of the 0.5-kbp BcII-HincII segment of the PHO80 gene (39) by the 1.4-kbp BamHI-XhoI HIS3 DNA fragment of S. cerevisiae. eStrain NBD81-6D, a pho8l null mutant, was a haploid segregant of the diploid strain NBD81 constructed by gene replacement of the 1.3-kbp BamHI-SmaI segment of the PHO81 gene (49) by the 1.4-kbp BamHI-XhoI HIS3 DNA fragment of S. cerevisiae.

siae chromosome as described by Orr-Weaver et al. (26). P-Galactosidase activities of colonies and of cell extracts of S. cerevisiae were measured as described before (49) and expressed as nanomoles of o-nitrophenyl-p3-D-galactoside cleaved per minute per milligram of protein. Protein concentration in cell extracts was determined with a protein assay kit (Bio-Rad Laboratories, Richmond, Calif.) with bovine serum albumin as a standard. General methods for the preparation, modification, and analyses of DNA and RNA were as described elsewhere (49, 50). Pi uptake was assayed as described previously (41). Preparation of a membrane fraction of yeast cells. Cells of S. cerevisiae were grown to the mid-log phase in low-Pi medium containing appropriate nutrients. The cells were collected, washed with SM buffer (20 mM Tris-hydrochloride buffer [pH 7.4] containing 85 mM NaCl and 1 mM MgSO4 7H20), and suspended at 2 x 109 cells per ml in lysis buffer (0.1 M Tris-hydrochloride buffer [pH 7.4] containing 20% [vollvol] glycerol, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Then, 0.5 g of glass beads per ml was added and the mixture was vigorously shaken in a microtube mixer MT360 (Tomy Seiko, Tokyo, Japan) at 4C for 30 min. The cell extract (crude extract) was centrifuged in an Eppendorf Microfuge for 15 min (10,000 x g) at 4C. The resulting supernatant fraction contained water-soluble protein, and the pellet fraction contained the total-membrane fraction (40). The pellet was suspended in an appropriate amount of the lysis buffer. Nucleotide sequence accession number. The nucleotide sequence reported has been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence data bases under the accession number D90346.
RESULTS Cloning the PH084 gene. Cloning of the PH084 gene was performed on the basis of its ability to suppress the pho84

mutant phenotype producing rAPase in high-Pi medium. A pho84 mutant, NS219 (pho3 pho84 ura3), was transformed with the S. cerevisiae genomic DNA library, YCp5O "CEN BANK" A, and Ura+ transformants were selected on SGly plates lacking uracil. A total of 12,000 Ura+ colonies that appeared on the plates were isolated and spotted on fresh SGly plates lacking uracil. The plates were incubated at 30C for 3 days, and the 12,000 Ura+ colonies that appeared on the plates were replicated onto fresh SGly plates lacking uracil. The plates were incubated at 30C for 2 days, and then the rAPase activities of colonies were examined by staining. Fifty colonies did not show rAPase activity. All of these clones were tested for rAPase production after they were cured of the plasmid by subculturing and streaking cells on YPAGly medium. One of them segregated only a class of clones showing red (rAPase+) Ura- phenotypes in the white (rAPase-) Ura+ population, while the other 49 clones segregated red Ura+ and white Ura- clones in addition to those showing the red Ura- phenotype. DNA was prepared from the former transformant and used to transform E. coli JA221 to Ampr. A plasmid obtained from one of the Ampr transformants conferred the rAPase- phenotype on yeast strain NS219 (pho3 pho84) in high-Pi medium. This plasmid, p373 (Fig. 1), had an insertion of a 7-kbp fragment in YCp5O. To determine whether the cloned DNA fragment carried the PH084 gene, we performed site-directed integration of the DNA fragment into an S. cerevisiae chromosome. A 5.6-kbp BamHI-EcoRI fragment of p373 was subcloned into the BamHI-EcoRI gap of YIp5. The resultant recombinant plasmid, pMB13 (Fig. 1), was linearized by restriction at the unique HpaI site in the cloned fragment. Strain NS216 (pho3 pho84 ura3) was transformed to the Ura+ phenotype with this DNA. The resultant transformants showed the wild-type phenotype for rAPase production, i.e., rAPase- in high-Pi medium and rAPase+ in low-Pi medium. We confirmed that the cloned fragment was integrated into the expected site of

VOL . 1 l, 1991

PHOSPHATE TRANSPORTER IN YEAST CELLS

3231

0~20
E 15

abC

tio

0.
0

a. 5

10 0

5 10 0 Time (Mnn)

10

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

FIG. 2. Pi uptake by the transformant cells of S. cerevisiae harboring plasmids connected with the PH084 gene. The transformant cells were shaken at 30C in synthetic low-Pi (0) or high-Pi (0) medium supplemented with appropriate nutrients without uracil. The cells were collected when cell growth reached an optical density at 660 nm of 1.0, washed, and inoculated into synthetic low-Pi medium to give a cell concentration with an optical density at 660 nm of 0.1. The radioactivity of the medium was adjusted to 5 x 105 cpm/ml with 32Pi. The amount of Pi absorbed by the cells was expressed as counts per minute of 32P radioactivity per milliliter in the cell suspension with an optical density at 660 nm of 0.1. Samples were taken at appropriate intervals and filtered through a nitrocellulose membrane filter. The test strains were MB191 (PHO84+) harboring pTI15 (a), MB192 (Apho84) harboring p373 (Fig. 1) (b), and MB192 harboring pTI15 (c).

FIG. 1. Structures and constructions of plasmids. The procedures for constructions of p373 and pMB13 are described in the text. pMB15 was constructed by ligation of a 5.6-kbp BamHI-EcoRI fragment of PH084 DNA prepared from p373 to the BamHI-EcoRI gap of pUC119. pMB93 was constructed by ligating a 7.8-kbp BstEII fragment of pMB15, after filling in with Klenow fragment, with a 1.7-kbp BamHI fragment containing the S. cerevisiae HIS3 sequence of YIpl, with filling in of the two ends. pMB123 was constructed by inserting the 1.7-kbp BamHI fragment of the HIS3 gene into the BglII site of pMB15 after elimination of a 2.0-kbp ApaI-BglII region and was connected with an 8-bp BglII linker (Takara Shuzo Co., Kyoto, Japan) at the ApaI end after digesting the overhanging 3' end to generate a blunt end with T4 DNA polymerase. pMB142 was constructed by inserting a 2.3-kbp HincII fragment of PH084 DNA, prepared from pMB15 and connected with a 10-bp BamHI linker, at the BamHI site of pSH540AB previously constructed in this laboratory for analysis of the HIS5 promoter (24). pMB143 was constructed by inserting the same 2.3-kbp HincIl fragment into the BamHI site of pMC1587 (4). The restriction sites on the plasmid DNAs are indicated only in the cloned PH084 fragment. Abbreviations of restriction sites: A, ApaI; B, BamHI; Bg, BglII; Bs, BstEII; C, ClaI; E, EcoRI; Hc, HincII; Hp, HpaI; N, NruI; S, Sau3AI; X, XhoI. S/B is the junction site of Sau3AI and BamHI.

the chromosome and that the integration was unique in the genome of three independent transformants by Southern hybridization of genomic DNA restricted with BglII and with 32P-labeled DNA of a 0.5-kbp BglII-XhoI fragment of pMB13 (Fig. 1) as a probe (data not shown). One of the clones in which it was integrated, MB159 (MATa pho3 pho84 ura3 leu2 trpl [PHO84' URA3+]), was crossed with AX49-4B (MATa pho3 his4 PHO84+), and the resultant diploid was subjected to tetrad analysis. Results showed an rAPase+/ rAPase- ratio of 0:4 on YPAD in 26 of the 28 asci dissected, the other 2 asci showing an rAPase+/rAPase- ratio of 1:3. These observations indicate that pMB13 DNA was integrated at or close to the pho84 locus and that the cloned DNA carried the PH084 gene. The effect of the plasmid bearing the PH084 gene on Pi transport was examined by measuring Pi uptake. Two strains, MB191 (PHO84+ ura3) and MB192 (pho84::HIS3 ura3) (see section on gene disruption of PH084 for construction of the pho84::HIS3 allele), isogenic to each other and harboring low-copy-number vector plasmid pTI15 (in MB191 and MB192) or p373 (in MB192) were assayed for Pi uptake activity (Fig. 2). MB192 carrying pTI15 did not take up Pi in either high-Pi or low-Pi medium. The transformant of MB191 with pTI15 and that of MB192 with p373 expressed Pi transport activity when incubated in low-Pi medium. Thus, the cloned PH084 gene also restored the Pi uptake activity of the pho84 mutant. Nucleotide sequence and mapping of the PH084 gene. For delimitation of the PH084 gene on the 5.6-kbp BamHIEcoRI fragment of PH084 cloned on pMB13, several deletion fragments were constructed from the cloned 7.0-kbp fragment (Fig. 3). pMB113, constructed by subcloning a 2.5-kbp ApaI-XhoI fragment from the 7.0-kbp fragment into the SmaI site of pTI15, suppressed the rAPase constitutive phenotype by the pho84 mutation, and pMB150, bearing the 4.7-kbp BamHI-XhoI fragment modified by restriction and

3232

BUN-YA ET AL.
BS2

MOL. CELL. BIOL.

BfIS
L- -3
Plasmid

bl

Bs3

Xb2

Sc Hc1DA

cHp,H

,2X
(

C3E,E22
lI
TUB3

S/B

Complementation

-2

-1

-2

-1

QI2(b)

(kbp)

etto

p373
pMB44

pMB109 pMB150 pMB113

pMB51

2-bp

FIG. 3. Restriction map of the DNA fragment cloned on p373 and its derivatives and their abilities to complement the rAPase constitutive phenotype of a pho84 mutant, NS219. The open and closed boxes at the top indicate the cloned yeast DNA. The closed box with an open arrow indicates the region in which the nucleotide sequences were determined. The open arrow labeled PH084 indicates the approximate position and direction of the ORF of PH084, and the open region labeled TUB3 indicates the N-terminal coding region of the TUB3 gene. Two or more identical restriction sites in the map are distinguished by subscripts. The lines indicate the PH084 fragments on the relevant YCp vectors. Plasmid pMB51 consisted of the indicated portion of the cloned fragment inserted at the ClaI site of YCp5O, and plasmids pMB113, pMB109, and pMB150 are subclones of the indicated portions in vector pTI15. pMB44 was constructed by deleting a 1.4-kbp XbaI fragment from p373. The symbols + and - indicate ability and inability of the plasmid to complement the pho84 mutant phenotype. Abbreviations of restriction sites are defined in the legend to Fig. 1, except for D (DraI), Sc (ScaI), and Xb (XbaI). None of the Dral sites are indicated, except for that at the end of the sequenced region of the PH084 DNA. The recognition site for ClaI indicated as C2 overlaps the methylation site of dam.

filling up of the ClaI site, did not complement the pho84 mutation. Therefore, we concluded that the 2.5-kbp ApaIXhoI region encodes the PH084 gene. A 3.9-kbp DraI-EcoRI fragment bearing the 2.5-kbp ApaIXhoI region (Fig. 3) was subcloned appropriately into pUC118 or pUC119, and its nucleotide sequence was determined by the dideoxy chain termination method. A single open reading frame (ORF) of 1,788 bp was found in the 3.9-kbp fragment (Fig. 4). Putative TATA boxes were found at nucleotide positions -122 to -118 and -99 to -95 in the 5' upstream region of the ORF relative to the translational initiation codon, while no sequences homologous to the terminator sequence, TAG. .TAGT. .TTT, proposed by Zaret and Sherman (51) were found in the 3' noncoding region. The GRFJO (= PH02 and BAS2 [50]) and PH04 genes are both required for transcriptional regulation of the PH05 gene (27, 48), and the bindings of PHO2 (44) and PHO4 (25, 44) proteins to the promoter region of PH05 were confirmed. Recently, Hayashi and Oshima (13) found that a 5-bp motif, 5'-CACGT-3', or preferably a 6-bp motif, 5'-CAC GTG-3', in the promoter region of the PH08 gene, encoding repressible alkaline phosphatase (EC 3.1.3.1), is essential for PH08 derepression and that the PHO4 protein binds to these motifs. In the upstream region of the PH084 DNA, we found three copies of the 5'-CACGTG-3' motif at nucleotide positions -880, -436, and -414 and a copy of the 5'-CACGTT3' motif at -587 (Fig. 4). No sequences homologous to those proposed for binding the GRF10 protein (2, 3, 37, 44) could be detected.

Comparison of the flanking region of the PHO84 ORF with the nucleotide sequence data of Schatz et al. (32) showed a part of the ORF of the TUB3 gene. This result was consistent with the results of subsequent conventional mapping analysis. The TUB3 gene is reported to show a linkage of 67 centimorgans (cM) to the GAL80 locus (33) on the left arm of chromosome XIII. We examined this by constructing a diploid by an MT8-1 (MATa leu2 gal8O::LEU2 PHO84+) x MB137 (MA Tc leu2 GAL80 pho84) cross. The tetrad data of 90 asci from the diploid indicated a similar result, 45-cM linkage, between the PHO84 and GAL80 loci with a 31:5:54 ratio for the parental ditype, nonparental ditype, and tetratype tetrads. The PHO84 and TUB3 loci are arranged in the same orientation, and the initiation codon of the TUB3 gene is only 355 bp from the termination codon of PHO84 (Fig. 4). These results indicate that the PHO84 and TUB3 loci are located side by side on the left arm of chromosome XIII. Structure of the predicted PHO84 protein. The predicted protein encoded by the PHO84 ORF contains 596 amino acid residues and has a molecular size of 65 kDa. By comparison with sequences in the data base of the European Molecular Biology Laboratory (Release 12.0; October, 1989) by using the GENETYX program (Software Development Co., Tokyo, Japan), we detected significant similarities of the predicted PHO84 protein with a glucose transporter of S. cerevisiae encoded by SNF3 (5). This SNF3 protein has been shown to be homologous with various mammalian, yeast, and bacterial sugar transporters (5, 35). The predicted PHO84 protein also has similarities with these other sugar transporters (Fig. 5). Hydropathy plots of the deduced amino acid sequences of the PHO84 and SNF3 proteins by the method of Kyte and Doolittle (17) also indicated significant similarities with these proteins, each consisting of 12 membrane-spanning segments connected by charged hydrophilic loops (Fig. 6). With the algorithm of Eisenberg (8), 12 putative membrane segments, each consisting of 21 amino acid residues, were identified in the PHO84 protein (overlined residues in Fig. 5). These putative membrane-spanning regions of the PHO84 protein were arranged into two groups, each consisting of six segments containing mainly hydrophobic amino acids and separated by a segment of 74 amino acid residues rich in charged amino acids (26 of the 74 amino acid residues in PHO84 protein and 22 of the 70 residues in the similar spacer segment of SNF3 glucose transporter). In addition to the above structural similarities, amino acid residues 63 to 297 in PHO84, covering segments 1 to 6, and residues 418 to 566 in segments 9 to 12 have significant homologies with the residues in the corresponding segments of SNF3, with 25% amino acid homology, and have similar levels of homology with those of corresponding regions in other sugar transporters, whereas the loop region and segments 7 and 8 have no significant amino acid homologies with those of SNF3 and other sugar transporters. The N-terminal 55 amino acids make up a large hydrophilic segment and so do not appear to constitute a signal sequence for insertion of the protein through the membrane. There is a potential threonine substrate site for cyclic AMP-dependent protein kinase in the PHO84 protein at amino acid position 558 with the sequence RKT. A potential N-linked glycosylation site (18) is also found at amino acid position 473 with the sequence NTT. Effect of gene disruption of PHO84. For examination of the effect of disruption of the PHO84 gene, pMB93 and pMB123 DNAs (Fig. 1) were doubly restricted with BamHI and EcoRI, and the resultant PHO84 fragments disrupted by insertion of the HIS3 DNA were used to transform cells of

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

VOL. 11, 1991

Oral
TTTAAAAAAGAAAAGA

PHOSPHATE TRANSPORTER IN YEAST CELLS

3233

I tit)

Cft

GGTTACTGGTTTACCTTAGATGTTGCTTTCTACGGGTTGAGTTTAAACAGTGCTGTTATTCTGCAAACCATCGGTTATGCCGGTTCCAAA
361 G Y N F D0 A F Y G 1 S L *1200 391N

-900

~~~~~~-850

GAAAAGGAATAAAAAAGTGTCACGTGATAAAAATCACTACCCGGAGATGACTTCAAACGACTCGGTATACTCTGCCTAATAAACCTTAAT * -800 -150 TTTCTTACAAAAAAAAAAAGATTCAATAAAAAAAGAAATGAGATCAAAAAAAAAAAAAATTAAAAAAAAAAAGAAACTAATTTATCAGCC *-100 -650 GCTCGTTTATCAACCGTTATTACCAAATTATGAATAAAAAAACCATATTATTATGAAAAGACACAACCGGAAGGGGAGATCACAGACCTT

* -550 ~~~~~~-600 GACCAAGAAAACATGCCAAGAAATGACAGCAATCAG TATTACGCACGT TGGTGC TGTTATAGGCGCCCTATACGTGCCAGCATIT TGCTCGTI -500 *u
I

TQ[D

[]S

A V I L 0 T I G Y A G S K

1250

AACGTTTACAAGAAACTGTATGATACTGCTGTCGGTAATCTGATTTTGATTTGTGCTGGTTCATTACCTGGTTACTGGGTATCCGTCTTC
YK K L YDT A V GN L IL ICA G SL P G Y V S OF * 1350 ~~~~~~~1300
K P IL AG F I ILTA LF CV IG F A Y

ACTGTCGATATAATCGGTAGAAAACCAATTCAATTAGCCGGTTTCATCATCTTGACCGCTTTGTTCTGTGTCATCGGTTTCGCATACCAT
421 T V DI I

OstEll

1400

AAGGGCCCTTTCAACTCATCTAGCGGCTATGAAGAAAATGTTGECCCGGCTGAAAAACACCCGTTCCTCTCACTGCCGCACCGCCCGATGC
-400

OAAACTTGGTGACCATGGTCTGTTGGCTCTTTACGTCATTTGTCAATTCTTCCAAAACTTCGGTCCAAACACAACCACCTTTATTGTTCCT
451 K L G 0 0 G L L A L Y V I C 0 F F 0 0 F G P N T T I F I V P 1450 bill1. 1500

-450

CAATTTAATAGTTCCACGTGGACGTGTTATTTCCAGCACGTGGGGCGGAAATTAGCGACGGCAATTGATTATGGTTCGCCGCAGTCCATC
*

GGTGAGTGTTTCCCAACTCGTTACAGATCTACTGCTCATGGTATTTCTGCTGCATCIGGTAAGGTCGGTGCCAITATTGCACAAACCGCT
CF P T RTYR STAONG ISA A SG KV GA I I A 481 G@E * 1550 OstEll 1600
TA

-350 -250

-300 -200

GAAATCAGTGAGATCGGTGCAGTTATGCACCAAATGTCGTGTGAAAGGCTTTCCTTATCCCTCTTCTCCCGTTTTGCCTGCTTATTAGCT

AGATTAAAAACGTGCGTATTAETCATTAATTAACCGACCTCATCTATGAGCTAATTATTATTCETTTITTGGCAGCATGATGEAACCACAT
*
*

TTGGGTACTCTAATCGACCATAACTGTGCTAGAGACGGTAAGCCAACCAACTGTTGGTTACCTCACGTCATGGAAATTTTCGCCTTATTC
5111L G I L I D H N C A R D G K P T N C N I P 0 V N E I F A L F O bal .1100 *1650 Cla'
54IN L LG IFTT L LI P 0OK RK TLEE IN E L YDE I * ~~~~~~1750 18000

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

~~~~-150

-100

TGCACACEGGTAATGCCAAETTAGATCCACTTACTATTGTGGCTCGTATACGTATATATATAAGCTCATCCTCATETCTTGTATAAAGTA
~~~~~~-50
*

ATGTTGTTGGGTATCTTCACAACCTTGTTGATCCCAGAAACTAAGAGAAAGACTCTAGAAGAAATTAACGAGCTATACCACGATGAAATC

AAGTTCTAAGTTCACTTCTAAATTTTATCTTTCCTEATCTCGTAGATCACCAGGGCAEACAACAAACAAAACTECACGAATACAATCCAA
*~~~~~s
50

ATGAGTTECGTCAATAAAGATACTATTCATGTTGCTGAAAGAAGTCTTCATAAAGAACAECTTACCGAAGGTGGTAACATGGCCTTCCAC
IOS S V NKD T I HVA ER SL 0K E NL TE G GN NA F 150 100 COal

GATCCTGCTACGCTAAACTTCAGAAACAAGAATAATGACATTGAATCTTCCAGCCCATCTCAACTTCAACATGAAGCATAAAAGCCTCAA
511 I P A T I N F 0 N K N N D I E S S S P S 0 L 0 0 E A * ~~~~~~~~~1850

AGATGCACTAAAACTTGTAAACTAGAACAAATAATACAAAAACATTTTTATAAACTTATTATCAAACCCCTTACATAATCTATAAATACT
1900
*

AACCATTTGAATGAITTTTGCTCATATTGAAGATCCTCTGGAAAGAAGAAGATTGGETTTGGAGTCCATCGATGACGAAGGITTTEGGTTGG
310 0 1 N I F A N I E D P 1 E 0 R R 1 A I E S I I 0 E G F G N * 200 250

1950

GTCAGGTTACATATTTATTCGATAATTTCTTTTAATTTCATTATTTCCTCACATCTCTCTGCCATCCTGTTGCTGGTGCCAGAGCAGAGC
2000

Xhol

2050 2150

CAACAAGTTAAGACEATCTCCATTGETGGTGTTGGTTTCTTGACAGATTCTTATGATATTTTTGCCATTAATTTGGGTATCACTATGATG
61 0 0 V K I I S I A G V G F 1 T I 5 Y D I F A I N I G I T N N 350 *300

ATATCGTCCTTTCTTTTITTAGTTCCAGACGTTACCCGACATATCATTTCTCGAGCCTCTGGaAAACCACGAAACGTTTTACAAATTGCACA
*2100
*

TCTAAAAGAAATATAAACACAGATCAGGTAICTCATAAAGTACATTAATCGACTAAGCAAGCGACTTGAGACAATGAGAGAGGTCATTAG

TCCTACGTTTACTGGCACGGTAGTATGECAGGTECAAGTCAAAECTTGTTGAAGGTTT'CACETTCTGTTGGTACTGTTATTGGTCAATTT
91 S Y V Y
N0 6 5
*

P G P 5 0 1 1 1 K V S I S V G T V I G 0 F ~~~~400 450

R E V015 2250 ~~~~~~2200 TATTAATGGTATGTATGCGTTCCTTTTTTTGTTCAATATTCGCAACCAATGGCACCTGTGGGACAGGGAAAGAAGTTTGATCTGATCTGG

1216G F G T I A

GGTTTTGGTACTTTAGCTGATATTGTTGGTCGTAAGAGAATTTATGGTATGGAACTTATTATCATGATTGTCTGTACCATTETGCAAACC I I 000R K 0 I Y G N E I I I N I V C T I 1 0 T ~~~~ ~~~500 AETGTTGCTCATTETECTGCTATTAACTTEGTTGCTCATTCTCCTGCTATTAACTTCGTTGCTGTTTTAACATTCTACCGTATTGTCATG

*

I N

*
2350
*

stEll

2300
2400

TTTGATTCATTCCCAATTGGTCACCATCTGGTTGATTTACGGCAAATAATTTGACTTGTACCAGCACAGTTTACTAAC0GTTTCTTTTTC

151 T V A 0 5 P A I N F V A H S P A I N F V A V L T F Y R 1 V N
550
600

TCCATTTTTTCTGGGCATACTCGGACGAAAAAGCTCATAATTGACCTCATTACATGGGGAGTGATTTTTGTGTCTTCTTCTTCGGAGGAT
2450

**2500
V GO0 A G CO C1 N A CNW EL

TGCTGGAACTTTTGTTATTTTTCTTTTTTACAACAGTTGGTCAAGCAGGTIGTCAAATAGGTAATGCATGCTGGGAATTGTACTCCCTAG
I81G IG I GG D Y PILS S I IT. SOF AIT TK W0060 I N G A 700 * 650

YSIL

*2550

2600

GTCTTTGCTAACCAAGCTTGGGGTCAAATCTCCGGTGGTATCATCGCTCTTATETTGGTTGETGETTACAAGGGCGAACTAGAATACGCA
211 V F A N 0 A N G 0 I 5 G G I I A I I I V A A Y K C E I E Y A
*

AGCATGGCATCAAGGAAGACGGCCATTTGGAGGATGGCTTGTCAAAACCTAAGGGAGGTGAAGAAGGATTTTCTACATTCTTCCATGAAA
E H G I1K E D GO IC L IDG I S K P K G G E K G F S I F FONE * ~~~~~~2650 Clal 2700

750

800

AACTCTGGTGCTGAATGTGATGCTAGATGTCAAAAGGCTTGTGACEAAATGTGGAGAATCETTATTGGGTTGGGTACCGTTCTAGGGTTG GCATGTTTGTATTTCAGATTAACTATTCCAGAATCTCCTAGATATCAATTGGATGTTAACGCTAAGTTGGAACTTGCTGCTGCCGCACAA R Y 0 1 I V N A K I E I A A A A 2710A C L Y F 0 1 1 ~~~~ ~~950 GAACAAGATGGCGAAAAGAAAATTCACGACACCAGTGATGAAGACATGGEAATTAACGGITTTGGAAAGAGCTTCTACTGCCGTCGAATCT

241 N 5 6 A E C D A R C 0 K A C I 0 N W R I L I G 1 G T V I G I 900 II * 0~~ ~~50 OjAc I I. .E

CGGGGTACGGAAAATTCGTCCCAAGAGCAATCTACGTGGATTTAGAGCCCAATGTTATCGATGAAGTACGTACAGGACGTTTCAAGGAGC TGOY G K FV P RA I TOO DL ECP NV I DE V RT G O F KE

* ~~~~~~~~~2750 TTTTCCATCCAGAACAATTGATTAACGGTAAGGAAGATGECGEEAATAAETACGCAAGAGGCCATTATACAGTGGGTAGAGAAATAGTGG L FON PE OL IN G K E D 0000N YA R G 00Y TV GR ElI V

IO(DEGOM

2800
*

2850

301 C 0 I 6 E K K I 0 I T S I E I N A I N 6 1 E R A 5 T A V C S 1000 1050

ATGAAGTTGAAGAAAGAATTAGAAAGATGGCCGACEAATGTGAEGGTTTACAAGGGTTCTTGTTCACCCACTCCCTCGGTGGTGGAACTG 0 EV E E R I RKX HAlOECD GILG G F I F 1051S G G GT

2900
..

EcoOl

ETTGACAATCATCCTECAAAGGETTCGTTEAAAGATTTCTGCAGACATTTTGGTCAATGGAAGTAECGGTAAGATTTTGCTAGGTACTGCT
331 L I N N P P K A S F K I F C R 0 F G 0 N K Y C K I I I G T A
FIG. 4.

GTTCCGGTTTAGGTTCCCTGTTATTAGAAAACTTATCGTATGAATACGGGAAGAAATCCAAATTGGAATTC
G S G L G SIL LL E N L S Y E Y G 8K K
K LE F

Nucleotide sequence and deduced amino acid sequence of the ORF of the PH084 gene. The indicated amino acid sequence is that

longest ORF found in the region sequenced. The TUB3 gene starts from the nucleotide at position 2144 relative to the ATG codon of PH084. The 808-bp TUB3 coding region contains a 298-bp intron, as described by Schatz et al. (32). Sequences homologous with the PH04 protein-binding motif 3'-CACGTG-5' or 3'-CACGTT-5' (13) and the putative TATA box are underlined. The 1 1 encircled amino acid residues, but not the 2 residues enclosed in squares, in the putative PH084 protein conform in position and identity with the 13 residues conserved in
17 sugar transporters

of the

(16).

dine

diploid strain NBW78 (his3lhis3 PHO84IPHO84) to histiprototrophy as described previously (31). We obtained
several

showed

2+ :2- ratio for the His

phenotypes, and all of the

These findare

His+

segregants showed the

that all of the

His'

transformants. Six of these

His'

transformants

ings indicate
zygous for

Pho84- phenotype. His+ transformants

hetero-

were

isolated,
were

at random. Three of them were

disrupted

with

fragment prepared from pMB93, and the other disrupted with that of pMB123. We confirmed that the PH084 locus of these clones was disrupted by Southern hybridization of genomic DNA digested with Hindlll with a 32P-labeled 1.4-kbp HindIII (at +644)-Xhol (+2029) fragment of the PH084 DNA as a probe for the clones disrupted with the fragment of pMB93 and a 1p labeled 2.0-kbp HincII (ca. -1400)-HindIII (+644) PH084 fragment as a probe for those disrupted with the fragment of pMB123. All six of these clones were subjected to tetrad analysis. Results in five to seven asci tested with each clone
three

the DNA

disruption is not lethal to the cell. We confirmed that the Apho84 allele was unable to complement the pho84-1 mutation by crossing one of the Apho84 segregants having the ot mating type with NS219 (MATa pho84-J). Regulation of PH084 expression. The low-Km, Pi transport
system
is
known
to
as

PHO84IApho84

alleles and that gene

be

under

the

control

of the

same

regulatory system

rAPase and to be

starvation (36, 41). For determination of

derepressed by Pi whether transcrip-

tion of the PH084 gene is controlled by the P1 concentration, Northern with

hybridizations of total RNAs prepared from cells disruption of the pho2, pho4, pho8O, pho8l, or pho84

3234

BUN-YA ET AL.

MOL. CELL. BIOL.

PH084 GAL2 SNF3 HGT XylE AraE

51 61 86 1 1 11

ME-PSSK-KL7IILMLANGGAVL M--NTQYNSS-YIFSIT--LVAT
IMi&

_ wDaF} D.PS 1-FG G GFVVQTD-FLRR1K-HIKDG-THY-SE-PPQKQSMMMSICVG.FVAV- FG F YDETG I HAKN PN-----D-I PAKVIEEYN--QTWVHRYGESI LP SLQ-F I GT--VESLN---T-VFVAPNLSE-SA aL-F I AGA--LP--FI--TDIIFV----L---T--TPRSLRrrRRMNMFV-SVAAA FG
ES

PK-KPMSfYVTVSLLCLCVR

I3FGWQQ8KTISI

G4Lj-

I (I I NF VAIP S QG PH084 101 GP-BT I IILSKG GR KGLSIVVSVYIVGII I[IASIN.--------FN1 A GAL2 116 -LSNV ISI R IIFSTIF FSIGN vGAGG-------LTAPFI SNF3 137 SFTA .----FVN R 60 TLTTLW-S I HGT A %I A wnvir tsvT Unfn If T A L-'lt;lYtII r*,ArIDC Pr) nc!r V TrrAftA EA r o AMIC_-T-P 1 UI A JL-ibur LA ALA II.bJNHI'lELUZ L lAAVLL U VSAW 'L-P rLUkrllNVUN XylE 53 A 58 --SRL-QEWX s AraE G }GLFN - eFRI GSgSUtAGAI LFVL GS IGSA---- FATS----4 5 RI IGIGIYPS II hY} i GAVFANQ GI I IA P11084 160 GAL2 172 --------KWYQYFI II IAVWPML I PKH LVSCYQLMITB I FLCTN IGI ISAVVPLYQ TIIKS IGAISTYQWAII LLVSSAVS SNF3 195 --------- ITL VI G HGT 119 II TTGFVPMYV AHI LVSFNQFAII FGL-LVYCV XylE 117 TVPVYLAG EVI RI I Y ASMQPMYI A. EN I AraE 112 ________- VASYTAPLY ISMYQLMVT --IVLAF I 6

CTFC V

GNSMLNULAFVSAVLMGFSKLGKS

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

VAHSPAINOV&F

----------FE

SFV

GTrL11QLGIVVGLIAQVFG

PH084
GAL2 SNF3 IIGT

XylE
AraE

227 230 252 176 182 166

293 275 297 220 232 210
358 334 359 275 281 262
422 387 412 328

CtYFRtI PSRYQ RI LIG O LV&IgELEYANSGAECDARCQKACDQ ----------------VL FAW MIGALTL ESPRYCJKYGTKSYSNSVQ4G SSFLAIGMFF ESPRY YV-LKj QGTI NDASS------------------SIIFIP4QCIVLPF ESPR L IIN ODSIMGN&--------------------DL FASECIP44tULL4 PESPR4MSRG NYF44SGDASWLNT------------Dc SD1FS--YS-GN----------------ALLALPAVLLIILVVF P4AEKG
N HPPKASFKDFC@lFGQWKIfl AQEQDGEKK HDT ED- INC PAV LI? IE GNASWGELF-STKT---KVFQR[4I 'RSI--AKSNYSPE JSEEjJKSLSF--- LRGV-PVHDSGLLEELI KeDYEASFGSSNFIDCFISSKSRPKQTLRMFT E@N4iSVL-KK-LRG---TAD--VTIIDL IKEESRQMMREKKVTILELFRSPA----YRnI I I KQE GIL-RK-IMG.-----NTLATQAVI K-HSLDHGR--K-TGGRL------LMFG

7-

P11084 GAL2 SNF3 HGT XylE

AraE

RIII

EVL-RM-LRD----TSEKAREEL

R-ESL---KLKQGGWALFKI
8

fN----VRRAVFSJ

PH08 4
GAL2 SNF3 IIGT

XylE
AraE

P11084 GAL2 SNF3 HGT

XylE
AraE

336 318

rLAFYGLSL4ILI AGSKNVYKKLYEOAVGNf,I -S YPGVFT IQLTGNNYFFYYGIKS L-------------DDSFETly IVVNFASTF ILQ aQFSGINFIFYYGVNFFNK V .-------------SNSfVSFITYAVNVVFNVPG Li1 AVVLQLSQQLSGINAVFYYOTSSFEK4V-------------QQPVYAgpgI(VNTAFT ----------- IAIIIVI I FTVLA WVMLS IfQF&INVVLYYAPEVFEE MYYAPFKM4 ---------FTEQQMIATLVVTFMFATFI giLLQAMQQFTGMNII 10 9 II IR I ---FC IHKEGD -------1IL-jLY QN]GP--N EN KCL ATMMACMVIYAS VTRLYPHGKSQPSSKGA NCMIVF- IFCYATTWA A ATWG IVIEFF &KVLV444VL-TIANFIVAI-VGCC -KTVAA ---AKV-MIAF A VGPG V RA TLI IAGM-AGCMILM1LT L-jQLPWMSYL----- SIVA FVRK I LGMAIGMFSLGT--F--YTQAP----- GIV-AILSM-LF-YVAAAMSWG V A RK LKI VMALGTLVLGYCLMQFDNGTASS-----GLSWLSVGM-TMMICIAGYAMSAA
FV
Y
_ _ _ _ _

P11084 474 TTT EPG qR :F AIWAQTALG IG-J..SAi GAL2 452 PVAWVIT F-- KSKCMALA W GFL----- IAFFPFI---G CT-IAANWLVN ----C SNF3 468 GVVWVIS

HGT XylE
AraE

385 389 378

537 504 526 437 448 437

PH084 GAL2 SNF3 HGT

PYI-VDTOS SLGAK IFF PVCWVLLS I AI PVVWhLC I K FGI-NWBSNfi---- IGATFLTLLDSINAAGTE3YTALiI Ng]- WIRRGNN.. HEA DFLN-e IIDEI CETLEEI MA-o3FM[>G . EGVKS EEI GC[LVAMFFYVFFF ET
PIPWFIVA L;QG

CER ES HN-CARD& K AINFYYGYV

[--AVAG WTSN 1----VGMCFQY- .VV-----EQLCG&MFII QWLANYF---- -VSWTFPMMDKNSWLVAHFHNGFSYWI L-- I

XylE AraE

SQSDKTPEEL pLGAEv or vgvnF FK GAs]sGaQG EE ----------PETKKTQQTATL ET KF Y.CMGV0A

AFV-G- I

KSSTGV VSPIAL rDPLQR.. ETK3LEERI WGSKAsGVSVY

FL I

KLMAGEKLRNIGV

FIG. 5. Sequence and structural homology of the predicted PH084 protein with various sugar transporters. PH084, predicted PH084 protein; GAL2, galactose transporter of S. cerevisiae (35); SNF3, high-affinity glucose transporter of S. cerevisiae (5); HGT, HepG2 glucose transporter of human cells (23); XylE and AraE, xylose and arabinose transporters of E. coli (19). The amino acid sequences of the proteins are aligned to obtain maximum fitting with the PH084 sequence. Residues identical in the PH084 protein and other proteins are boxed. The lines above the PH084 sequences with numbers from 1 to 12 indicate putative membrane-spanning segments with 21 amino acid residues deduced from the hydropathy plot shown in Fig. 6. The numbers on the left represent the positions of the first amino acid residues of the indicated protein. Dashes indicate gaps introduced to optimize the alignment.

VOL . 1 l, 1991

PHOSPHATE TRANSPORTER IN YEAST CELLS

3235

3a
9

+ -7 _Ir +

a I- _ls_,+_ls_

<

<

<

PH084-

ACT1

** ** * 0 ft

*-2.3 kb

- -1.2 kb

0020300

400

500

600

700

800

1 2 3 4 5 6 7 8 9 1011 12 1314 FIG. 7. Detection of the PHO84 transcript by Northern hybridization. Total RNAs were prepared from cells of strains P-28-24C (wild type [WT]), NBD2-1 (Apho2), NBD4-1 (Apho4), NBD80-1 (Apho8O), NBD81-6D (Apho8J), MB192 (Apho84), and NS219 (pho84-1) grown on nutrient high-Pi (+) or low-Pi (-) medium. Samples (10 ,ug) of total RNA were used. After electrophoresis, the lanes were hybridized with a 32P-labeled 0.7-kbp ClaI-HpaI fragment of the PHO84 DNA and a 1.0-kbp XhoI-HindIII fragment of S. cerevisiae encoding the ACTI gene as probes. The specific activities of both probes were 1.0 x 108 cpm/,ug of DNA. 25S and 18S rRNAs visualized by staining with ethidium bromide were used as size markers as described by Philippsen et al. (29).

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

FIG. 6. Hydrophobicity profiles of the predicted PHO84 and SNF3 proteins. Hydropathy values for a window of 21 amino acid residues were averaged, assigned to the middle residue of the span, and plotted with respect to position along the amino acid sequence. The numbers refer to putative membrane-spanning domains predicted by the algorithm of Eisenberg (8).

and from the wild-type strain cultivated for 12 to 16 h (stationary phase) in nutrient low-P1 and high-Pi media were performed with a 32P-labeled 0.7-kbp ClaI-HpaI fragment of the PH084 DNA (Fig. 3) and a 1.0-kbp XhoI-HindIII fragment of the ACT] gene of S. cerevisiae (10) as probes. The RNA of the wild-type strain cultivated in low-Pi medium gave one hybridization band of approximately 2.3 kb, but that of cells cultivated in high-Pi medium gave no band (Fig. 7). This 2.3-kb PHO84 transcript is consistent in size with the PHO84 coding region. The pho2, pho4, and pho8l disruptants did not produce the PHO84 transcript, while the pho8O disruptant showed the same level of derepression of PHO84 transcripts in high-Pi and low-Pi media. The pho84 disruptant did not produce the transcript in either high-Pi or low-Pi medium. In contrast, cells of the pho84-1 mutant showed a low but significant level of transcription of the pho84-1 gene in high-Pi medium and a level in low-Pi medium similar to that in the wild-type cells (Fig. 7, lanes 13 and 14). These differential transcriptions of the pho84-1 gene in the pho84-1 mutant in high-Pi and low-Pi media are comparable with the differential rAPase synthesis of the same mutant (42). Like the pho8O disruptant, a pho85-1 mutant, YAT965 (49, 50), transcribed the PHO84 gene at similar levels in high-Pi and low-Pi media (data not shown). These results clearly indicate that PHO84 transcription is under the control of the PHO regulatory system. To confirm that PHO84 expression is under the control of Pi in the medium, we examined the expression of a PHO84lacZ fusion gene with pMB142 (Fig. 1). The 2.3-kbp HincIl fragment of the PHO84 DNA has a 1.4-kbp upstream region and a 867-bp ORF region encoding the N-terminal half of the PHO84 protein (Fig. 3 and 4). Though the PHO84 ORF was truncated, the PHO84-lacZ fusion gene has a sufficient
gene

region of the PH084 promoter, and the N-terminal coding region of PH084 was connected with lacZ in frame, because colonies of S. cerevisiae DKD-5D harboring pMB142 gave an intense blue color on plates containing the low-Pi version of SD medium on addition of 5-bromo-4-chloro-3-indolyl-3D-galactoside (X-Gal; Wako Pure Chemical Industries, Osaka, Japan). When the transformant cells were cultivated on high-Pi or low-Pi medium to the stationary phase, the activity profiles of P-galactosidase showed derepression by Pi starvation (Fig. 8). Thus, expression of the PHO84 ORF is under the control of P1 and is derepressed by P1 starvation. Cellular localization of PHO84-,-galactosidase chimeric protein. For determination of the location of PHO84 protein in S. cerevisiae cells, a PHO84-p-galactosidase chimeric protein which has 289 amino acid residues containing six transmembrane regions of the N-terminal half of the PHO84 protein connected with ,-galactosidase was produced in cells of NBW5 harboring pMB143 (Fig. 1). As a control in this analysis, soluble ,-galactosidase activity was examined in the same S. cerevisiae strain, NBW5, harboring plasmid pSH530 (a YEp-type plasmid similar to pMB143) carrying a HIS5-lacZ fusion gene that produces a fused protein consisting of the N-terminal 40 amino acid residues of the HIS5 protein connected with ,-galactosidase (24). The transformant cells were cultivated in nutrient low-Pi medium at 30C for 16 h and then harvested, washed, and disrupted, and their supernatant and membrane (sediment) fractions were prepared. We found that the total-membrane fraction prepared from the transformant with pMB143 contained high P-galactosidase activity whereas most of the 3-galactosidase activity of cells harboring pSH530 was in the soluble fraction (Table 2). These results suggest that the 289 N-terminal amino acid residues of the PHO84 protein function in transfer of the chimeric protein to the membrane. DISCUSSION The PHO84 gene of S. cerevisiae has been cloned by complementation of the constitutive phenotype of rAPase of the pho84 mutant. The following findings in this work

3236

BUN-YA ET AL.

MOL. CELL. BIOL.

0~~~~~~~~

50
0

0.01
24 16 Time (h) FIG. 8. Time courses of change in ,8-galactosidase activity in transformant harboring the PHO84-lacZ gene-fusion plasmid, pMB142. Cells of the transformant of DKD-5D harboring plasmid pMB142 were precultivated at 30C for 1 day in 5 ml of synthetic high-Pi medium supplemented with appropriate nutrients but without tryptophan. The cells were harvested, washed with sterile water, and inoculated into 100 ml of nutrient high-Pi (open symbols) or low-Pi (closed symbols) medium to give an optical density at 660 nm (OD660) of 0.01. The cultures were shaken gently at 30C and assayed for ,B-galactosidase activity (E and A) (in units per milligram of protein). Cell growth (O and *) was monitored as the OD.6 of the cultures. 0

strongly suggest that PHO84 encodes a Pi transport protein: the amino acid sequence deduced from the nucleotide sequence of the gene is highly homologous with those of several sugar transporters of mammalian, yeast, and bacterial origin (Fig. 5), and the P-galactosidase activity expressed by the PHO84-lacZ fusion gene seemed to be associated with the membrane (Table 2). The glucose transporters of S. cerevisiae, SNF3 (5), and HepG2 erythrocytes (HGT [23]), the xylose and arabinose transporters of E. coli (XylE and AraE [19]), and the maltose (MAL61 [6]) and galactose (GAL2 [351) transporters of S. cerevisiae have been shown to be homologous, and all of these sugar transporters have been grouped into an HGT superfamily (5, 35). These proteins all have 12 membranespanning regions consisting of two blocks of six transmembrane domains separated by charged, hydrophilic connecting loops. Duplicated GRR sequences are conserved in membrane-spanning segments 2 and 8, and PESPR and PETK sequences are conserved in membrane-spanning segments 6 and 12, respectively, in the HGT superfamily (35). The PHO84 protein also has these sequences starting at amino
TABLE 2. ,B-Galactosidase activity in membrane and supernatant fractions
Plasmid

acid positions 130, 280, 426, and 552 (Fig. 4 and 5). In human glucose transporters, the polar amino acid groups in segments 7 and 8 have been suggested to be located on one surface of the ct-helix and thus to form a polar channel through which the sugar is transported (11). Transporters of the HGT superfamily show high conservation of segments 7 and 8, but segment 7 of the PHO84 protein shows little homology with those of the HGT superfamily. Recently, 13 residues with perfectly conserved homology and position were found in 17 sugar transporters of various origins (16). In the putative PHO84 protein, 2 of these 13 conserved residues in segment 7 are different but the other 11 are conserved (Fig. 4). In addition, a tryptophan residue in segment 11 (amino acid position 412) of HGT is suggested to be a potential cytochalasin B-binding site (45), and this tryptophan residue is conserved in these sugar transporters but not in the PHO84 protein, in which the corresponding amino acid residue is lysine (Fig. 5). These differences might be because the PHO84 protein is specific for Pi but not sugar transportation. However, it is strange that the amino acid sequence and predicted structure of the PHO84 protein differ significantly from those of the putative Pi transporter of Neurospora crassa encoded by pho4+, which has 12 possible membrane-spanning segments with a loop region between segments 8 and 9 (20), and that of rat liver, which has 6 possible membrane-spanning segments (9). Ambudkar et al. (1) suggested that secondary carriers having 6 membranespanning segments in the molecule function as dimers and that those with 12 segments function as monomers. If this is so, the PHO84 Pi transporter may function as a monomer. pho84 disruption was not lethal but almost abolished Pi uptake (reference 41 and Fig. 2). This might be due to the fact that there are at least two systems of Pi transport in S. cerevisiae: one has high affinity for external Pi and is repressed by Pi, and the other is constitutive and has low affinity to Pi (36). The Pi transporter encoded by PH084 corresponds to the repressible system. It is unknown whether the PHO84 protein contributes to the constitutive system. Northern blot hybridization studies showed that PH084 transcription is regulated by the Pi concentration in the medium and is under the same transcriptional control as the PH05 and PHO81 genes (Fig. 7). Four copies of the 5'CACGTG/T-3' motif, which is essential for derepression of the PH08 gene (13), were found at positions -880, -587, -436, and -414.
ACKNOWLEDGMENTS We thank T. Fukasawa and Y. Suzuki of Keio University for providing the gal80 disruptant and D. Gallwitz of Max-PlanckInstitute for the pYA301 plasmid. We also thank H. Saitoh of our laboratory for technical assistance. This study was supported by Grants-in-Aid for Special Project Research and for General Scientific Research to Y.O. from the Ministry of Education, Science, and Culture of Japan.
REFERENCES 1. Ambudkar, S. V., V. Anantharam, and P. C. Maloney. 1990. UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer. J. Biol. Chem. 265:12287-12292. 2. Arndt, K. T., C. Styles, and G. R. Fink. 1987. Multiple global regulators control HIS4 transcription in yeast. Science 237:874880. 3. Braus, G., H.-U. Mosch, K. Vogel, A. Hinnen, and R. Hutter. 1989. Interpathway regulation of the TRP4 gene of yeast. EMBO J. 8:939-945. 4. Casadaban, M. J., A. Martinez-Arias, S. K. Shapira, and J.

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

pSH530 pMB143

extractb Membranes Supernatant 65 + 17 1,090 + 150 HIS5-lacZ 1,130 + 260 PH084-lacZ 2,160 + 250 1,880 + 246 221 + 82
Cell

Gene fusion

,-Galactosidase activitya in:

a In units per milligram of protein + standard deviation. Values are means for triplicate determinations. b Unfractionated cell extract.

VOL . 1l, 1991

PHOSPHATE TRANSPORTER IN YEAST CELLS

3237

5.

6.

7.

8.

9.

10.
11. 12.

13.

14.

15.

16.
17.

18. 19.

20.

21.
22. 23.

24.

25.

Chou. 1983. P-Galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol. 100:293-308. Celenza, J. L., L. Marshall-Carlson, and M. Carlson. 1988. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc. Natl. Acad. Sci. USA 85:21302134. Cheng, Q., and C. A. Michels. 1989. The maltose permease encoded by the MAL61 gene of Saccharomyces cerevisiae exhibits both sequence and structural homology to other sugar transporters. Genetics 123:477-484. Clarke, L., and J. Carbon. 1978. Functional expression of cloned yeast DNA in Escherichia coli: specific complementation of argininosuccinate lyase (argH) mutations. J. Mol. Biol. 120:517-532. Eisenberg, D. 1984. Three-dimensional structure of membrane and surface proteins. Annu. Rev. Biochem. 53:595-623. Ferreira, G. C., R. D. Pratt, and P. L. Pedersen. 1989. Energylinked anion transport: cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial protein/phosphate symporter. J. Biol. Chem. 264:15628-15633. Galiwitz, D., and I. Sures. 1980. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 77:2546-2550. Gould, G. W., and G. I. Bell. 1990. Facilitative glucose transporters: an expanding family. Trends Biochem. Sci. 15:18-23. Harashima, S., A. Takagi, and Y. Oshima. 1984. Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol. Cell. Biol. 4:771-778. Hayashi, N., and Y. Oshima. 1991. Specific cis-acting sequence for PH08 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:785-794. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. Kaneko, Y., A. Toh-e, and Y. Oshima. 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. Kruckeberg, A. L., and L. F. Bisson. 1990. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport. Mol. Cell. Biol. 10:5903-5913. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. Lehle, L., and E. Bause. 1984. Primary structural requirements for N- and O-glycosylation of yeast mannoproteins. Biochim. Biophys. Acta 799:246-251. Maiden, M. C. J., E. 0. Davis, S. A. Baldwin, D. C. M. Moore, and P. J. F. Henderson. 1987. Mammalian and bacterial sugar transport proteins are homologous. Nature (London) 325:641643. Mann, B. J., B. J. Bowman, J. Grotelueschen, and R. L. Metzenberg. 1989. Nucleotide sequence of pho4+, encoding a phosphate-repressible phosphate permease of Neurospora crassa. Gene 83:281-289. Morrison, D. A. 1977. Transformation in Escherichia coli: cryogenic preservation of competent cells. J. Bacteriol. 132: 349-351. Mortimer, R. K., D. Schild, C. R. Contopoulou, and J. A. Kans. 1989. Genetic map of Saccharomyces cerevisiae, edition 10. Yeast 5:321-403. Mueckler, M., C. Caruso, S. A. Baldwin, M. Panico, I. Blench, H. R. Morris, W. J. Allard, G. E. Lienhard, and H. F. Lodish. 1985. Sequence and structure of a human glucose transporter. Science 229:941-945. Nishiwaki, K., N. Hayashi, S. Irie, D.-H. Chung, S. Harashima, and Y. Oshima. 1987. Structure of the yeast HIS5 gene responsive to general control of amino acid biosynthesis. Mol. Gen. Genet. 208:159-167. Ogawa, N., and Y. Oshima. 1990. Functional domains of a positive regulatory protein, PHO4, for transcriptional control of

26. 27.

28. 29.

30.

31. 32.

33.

34.

35.
36. 37. 38.

39.
40.

41.

42.

43.
44.

the phosphatase regulon in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:2224-2236. Orr-Weaver, T. L., J. W. Szostak, and R. J. Rothstein. 1981. Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78:6354-6358. Oshima, Y. 1982. Regulatory circuits for gene expression: the metabolism of galactose and of phosphate, p. 159-180. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces: metabolism and gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Parent, S. A., C. M. Fenimore, and K. A. Bostian. 1985. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1:83-138. Philippsen, P., M. Thomas, R. A. Kramer, and R. W. Davis. 1978. Unique arrangement of coding sequences for SS, 5.8S, 18S and 25S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis. J. Mol. Biol. 123:387-404. Rose, M. D., P. Novick, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237243. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-211. Schatz, P. J., L. Pillus, P. Grisafi, F. Solomon, and D. Botstein. 1986. Two functional a-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins. Mol. Cell. Biol. 6:3711-3721. Schatz, P. J., F. Solomon, and D. Botstein. 1986. Genetically essential and nonessential a-tubulin genes specify functionally interchangeable proteins. Mol. Cell. Biol. 6:3722-3733. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Szkutnicka, K., J. F. Tschopp, L. Andrews, and V. P. Cirillo. 1989. Sequence and structure of the yeast galactose transporter. J. Bacteriol. 171:4486-4493. Tamai, Y., A. Toh-e, and Y. Oshima. 1985. Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae. J. Bacteriol. 164:964-968. Tice-Baldwin, K., G. R. Fink, and K. T. Arndt. 1989. BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2. Science 246:931-935. Toh-e, A., and Y. Oshima. 1974. Characterization of a dominant, constitutive mutation, PHOO, for the repressible acid phosphatase synthesis in Saccharomyces cerevisiae. J. Bacteriol. 120:608-617. Toh-e, A., and T. Shimauchi. 1986. Cloning and sequencing of the PHO80 gene and CEN15 of Saccharomyces cerevisiae. Yeast 2:129-139. Tschopp, J. F., S. D. Emr, C. Field, and R. Schekman. 1986. GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J. Bacteriol. 166:313318. Ueda, Y., and Y. Oshima. 1975. A constitutive mutation, phoT, of the repressible acid phosphatase synthesis with inability to transport inorganic phosphate in Saccharomyces cerevisiae. Mol. Gen. Genet. 136:255-259. Ueda, Y., A. Toh-e, and Y. Oshima. 1975. Isolation and characterization of recessive, constitutive mutations for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. J. Bacteriol. 122:911-922. Vieira, J., and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3-11. Vogel, K., W. Horz, and A. Hinnen. 1989. The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PH05 upstream activation regions. Mol. Cell. Biol.

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham

9:2050-2057.
45. Walmsley, A. R. 1988. The dynamics of the glucose transporter. Trends Biochem. Sci. 13:226-231. 46. Wickner, R. B. 1987. MKTJ, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replica-

3238

BUN-YA ET AL.

MOL. CELL. BIOL.

tion of M2 double-stranded RNA. J. Bacteriol. 169:4941-4945. 47. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119. 48. Yoshida, K., Z. Kuromitsu, N. Ogawa, K. Ogawa, and Y. Oshima. 1987. Regulatory circuit for phosphatase synthesis in Saccharomyces cerevisiae, p. 49-55. In A. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E. Yagil (ed.), Phosphate metabolism and cellular regulation in microorganisms. American Society for Microbiology, Washington, D.C.

49. Yoshida, K., Z. Kuromitsu, N. Ogawa, and Y. Oshima. 1989. Mode of expression of the positive regulatory genes PH02 and PH04 of the phosphatase regulon in Saccharomyces cerevisiae. Mol. Gen. Genet. 217:31-39. 50. Yoshida, K., N. Ogawa, and Y. Oshima. 1989. Function of PHO regulatory genes for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 217:40-46. 51. Zaret, K. S., and F. Sherman. 1982. DNA sequence required for efficient transcription termination in yeast. Cell 28:563-573.

Downloaded from http://mcb.asm.org/ on January 10, 2013 by Univ of Nottingham