- Email: [email protected]
A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae
Gene, 171 (1996) 41-47 ©1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00
41
GENE 09677
A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae (Phosphatase regulation; Pi transporter; yeast)
Chulee Yompakdee*, Masanori Bun-ya*'**, Koh Shikata, Nobuo Ogawa, Satoshi Harashima and Yasuji Oshima Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka Suita-shi, Osaka 565, Japan Received by J. Marmur: 9 November 1995; Revised/Accepted: 29 November/5 December 1995; Received at publishers: 26 January 1996
SUMMARY
The PH084 gene in Saccharomyces cerevisiae encodes the Pi transporter Pho84p. The other three genes, GTR1, PH086 and PH087, are also suggested to be involved in the Pi uptake system. We cloned and sequenced PH086 and found that it encodes a 34-kDa protein consisting of 311 amino acid residues with two strongly hydrophobic segments in its N-terminal half. Western blotting analysis of cell extracts revealed that Pho86p, tagged with c-Myc, was fractionated into a water-insoluble fraction. Disruption of PH086 did not affect cell viability even in combination with the pho84 and/or pho87 disruptions. The triple disruptants showed high levels of constitutive rAPase synthesis and arsenate resistance similar to the pho84 mutant, but showed slower cell growth than the pho84 mutant. PH086 has two putative binding sites for the transcriptional activator, Pho4p, at nucleotide positions - 1 9 1 and - 4 9 7 relative to the ATG start codon, and showed substantial levels of transcription under high-Pi conditions and more enhanced levels in low-P~ medium.
INTRODUCTION
Two systems are suggested to operate in active transport of Pi in Saccharomyces cerevisiae (Sc; Tamai et al., 1985). One has a low Km value (8.2 laM) for external Pi, and the other has high Km value (770 ~tM). PH084 has been shown to encode a major component of the Pi transporter for the low Km system (Bun-ya et al., 1991). PH084 transcription is under the stringent control of Pi concentration in the medium through the same system Correspondence to: Dr. Y. Oshima, Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan. Tel. (81-6) 879-7420; Fax (81-6) 879-7448; e-mail [email protected] * The first and the second authors contributed equally to this work. ** Present address: Faculty of Integrated Arts and Sciences, Hiroshima University, Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 724, Japan. Tel. (81-824) 24-6564. Abbreviations: aa, amino acid(s); bp, base pair(s); A, deletion; E.,
PII S0378-1119(96)00079-0
as for regulation of repressible acid phosphatase (rAPase; EC 3.1.3.2) encoded by PH05 (for review, see Johnston and Carlson, 1992). The PHO regulatory system responsive to the Pi signals consists of products of at least five genes, PH02 (=BAS2/GRFIO), PH04, PH080, PH081 and PH085. A transcriptional activator, Pho4p encoded by PH04, binds to the promoter region of the PHO structural genes. Pho2p is another transcriptional activator in the PHO genes, except PH08 encoding repressible alkaline phosphatase (EC 3.1.3.1) in vacuole, and is effective Escherichia; GTRI, gene encoding Gtrlp; Gtrlp, putative GTP-binding protein; kb, kilobase(s) or 1000 bp; MCS, multiple cloning site; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; PH04, gene encoding transcriptional activator Pho4p in the PHO reguIon; PH084, gene encoding the Pi transporter Pho84p; PH086 and PH087, genes encoding Pho86p and Pho87p, respectively, putative members of the Pi transporter complex; Pi, inorganic phosphate; rAPase, repressible acid phosphatase; Sc, Saccharomyces cerevisiae;wt, wild type; YPAD, yeast extract peptone adenine dextrose (medium); YPAD-P, YPAD medium with low Pi; : :, insertional disruption of a gene.
42 for transcription of several genes for synthesis of various aa and adenine. In high-Pi medium (containing 11.0 mM or more Pi), Pho80p (a kind of cyclin) and Pho85p (cyclin-dependent protein kinase) form a complex which inhibits Pho4p function by hyperphosphorylation of it (Kaffman et al., 1994). Then the cells are unable to enhance the transcription of PH05 and the other genes in the PHO regulon. In low-Pi conditions (0.22 mM or less P0, Pho81p inactivates the Pho80p-Pho85p complex (Schneider et al., 1994; Ogawa et al., 1995a), then Pho4p is active for transcription of the PHO genes. Transcription of PH081 is also regulated by Pi via the same PHO regulatory system (Ogawa et al., 1993). Thus, the PHO regulatory system forms a positive feedback loop. A pho84 mutation confers a rAPase + phenotype, irrespective of P~ concentration in the medium, possibly due to reduction of intracellular P~ concentration or disturbance in a Pi signal transduction mechanism. In a previous work (Bun-ya et al., 1996), we isolated a pho86 pho87 double mutant, defective in Pi uptake, by isolating mutants showing constitutive synthesis of rAPase and arsenate resistance, speculating that arsenate functions as an analog of P~ in many cellular reactions (Willsky et al., 1985) and inhibits cellular metabolism and cell growth. The single pho86 mutant, however, showed only moderate phenotypes of the double mutant and the single pho87 mutant was indistinguishable from the wt strain. Thus, PH087 plays a cooperative but auxiliary role in conjunction with PH086 in the P~-uptake system. PH087 is identical to YCR524, which was detected in the course of a Sc chromosome sequencing project (Thierry et al., 1990). The published nt sequence of PH087/ YCR524 showed that it encodes a putative 923-aa (103-kDa) membrane protein (Bun-ya et al., 1996). The aim of present studies was to characterize the PH086 gene. We found that it encodes a putative 3 l l-aa
membrane protein with two strongly hydrophobic segments in its N-terminal half. Its transcription was suggested to be under the relaxed control of Pi. Disruption of PH086 was not lethal to the cells.
RESULTS AND DISCUSSION
(a) Cloning and sequencing of PH086 To clone the PH086 gene, strain NS213 (pho86 pho87 ura3; Table I) was transformed to the Ura + phenotype with a gene library, 'CEN BANK' A (obtained from the American Type Culture Collection, Roekville, MD, USA), constructed by partial digestion of Sc DNA with Sau3AI, and ligation with YCp50 (marked with URA3; Parent et al., 1985) at the unique BamHI site. We selected three clones showing the rAPase- phenotype by staining approximately 4000 Ura ÷ colonies on high-Pi plates according to the plate assay method of rAPase as described previously (Bun-ya et al., 1991). Then, the plasmid DNAs of these Ura + rAPase- transformants were analyzed after propagation in E. coli. We found that each of these three plasmids contains an identical 4.5-kb insert (designated pMB216; Fig. 1) and confirmed that the insert is PH086 by integration of it into a homologous site in a chromosome using an integrative vector, YIp5 (Parent et al., 1985), followed by tetrad analysis of the transformant (details not shown). The restriction map (Fig. 1) of the insert apparently differed from PH084 (Bun-ya et al., 1991) and PH087 (Bun-ya et al., 1996). The essential region for the PH086 activity was narrowed down to a 2.1-kb SphIz-SpeI1 region (Fig. 1). Sequence determination of a 3-kb SphIi-EcoRV fragment (the EcoRV site is on the vector moiety beside the Sau3AI/BamH1 ligation site) revealed an ORF of 933 bp, encoding a 34-kDa protein with 311 aa residues (Fig. 2),
TABLE I Yeast strains Strain
Genotype a
Source or reference
P-28-24C NS213 SH3319 b SH3417 SH3418 NBD8167-1A c NBD8687dl c
MATa MATa MATer MATer MATa MATa MATs
Our stock Bun-ya et al. (19961 This study Bun-ya et al. (1996) Bun-ya et al. (1992) This study This study
pho3-1 pho3-1 pho86-1 pho87-1 leu2 trpl ura3 pho3-1 pho5-1 gcn4-103 pho4::LEU2 hisl-29 leu2-3, 112 ura3-52 [HIS4-1acZ, ura3-52] ura3 leu2 trpl his3 pho87::URA3 pho84::HIS3 his3-532 leu2 trpl ura3-1,2 pho3-1 leu2-3, 112 ura3-1,2 trpl-289 his3-532 ade2 canl pho86::HIS3 pho3-1 leu2-3, 112 ura3-1,2 trpl-289 his3-352 ade2 canl pho86::HIS3 pho87::URA3
a The genetic symbols used are as described by Mortimer et al. { 19921. b A derivative of strain SH3071 [PH05 ÷]Apho4 (Harashima et al., 19951 ¢ Strain NBD8687dl, a Apho86 Apho87 disruptant, was constructed from a derivative of NBW7 (Ogawa and Oshima, 1990) having the pho87::URA3 allele (Bun-ya et al., 1996) by replacement of the 1.1-kb BamHI~-Pstl segment of PH086 (Fig. 1) with a 1.2-kb BamHI-PstI HIS3 fragment of Sc prepared from YIpl (Parent et al., 19851. Strain NBD8167-1A is a haploid segregant from a diploid of the NBD8687dl x NOG1 (a derivative of NBW7t cross.
43 Pvl h S 1
E1 Sh2
Xb2 H1
B
He~JHp/A2 Pv2 A1 Spl H2 B Pv P Sa/BEV
INCA3
-,2
Plasmid
?
I
~(~) Complementation
+
pMB216 pMB217 pMB220 pKS15 pMB221 pKS8 pKS9 pKS10 pKS13
1
Chr.IV
+ J [
LEU2
I
m m
m
•
+
Fig. 1. Restriction map of the PH086 DNA and delimitation of the effective region for the PH086 function. The open and closed boxes indicate the cloned Sc DNA and the thin line the YCp50 DNA. Complementation of the DNA fragments was examined by suppression of the rAPase+ phenotype in NS213 (pho86 pho87) cells on high-Pi (YPAD) medium. The symbols + and - indicate complementation ability and inability, respectively. Abbreviations of restriction sites: A, AccI; B, BamHI; E, EcoRI; EV, EcoRV; H, HindllI; Hc, HinclI; P, PstI; Sh, SphI; Sp, SpeI; Pv, PvulI; Xb, XbaI; B/Sa, BamHl/Sau3AI; and Hc/Hp/A, HinclI/HpaI/AccI. Methods:pMB216 is the original plasmid in which the PH086 DNA was cloned on YCp50 plasmids pMB217, pMB220 and pMB221 were constructed by deleting a HindlII, SphI or XbaI fragment, respectively, from pMB216, pKS8 was constructed by eliminating a 221-bp BamHI fragment from pMB220. A 2.7-kb HindlIf~-EcoRV fragment of PH086 from pMB216 was subcloned into the HindlII-HinclI gap of pUCll9 (Sambrook et al., 1989), and the resulting plasmid was named pMB230 (not shown), pKS9 and pKS10 were constructed by inserting a 2.0-kb BamHIz-BamHl (the latter BamHI site exists on the pUC119 moiety) and a 1.2-kb HindlII1-HinclI2 fragment of PH086 from pMB230 into the BamHI site or the HindllI-NruI gap of YCp50, respectively.A 0.6-kb SpeI segment was removedfrom pMB230, and a 2.1-kb HindllIl-EcoRl (EcoRI site exists on the vector) fragment containing the SpeI deletion was inserted into the 2.7-kb HindlIIi-EcoRI gap of pMB220 to generate pKSI3. The PH086 function of the cloned fragments was examined by complementation of the rAPase÷ phenotype of strain NS213 (pho86 pho87 ura3) on high-Pi medium to the rAPase- phenotypeby introducing the plasmids (marked with URA3 bearing the fragment to be tested) into the NS213 cells.
in the 2.1-kb essential SphI2-SpeI a region. Thus, we concluded that this O R F encodes Pho86p. We also found a part of another O R F in the 3-kb sequenced fragment in tail to tail arrangement with the P H 0 8 6 ORF, but it was located outside of the 2.1-kb essential region (Fig. 1). In a search of the nt and aa sequences in the GenBank database, we found that P H 0 8 6 encodes a unique protein, and that the neighboring partial O R F is identical to N C A 3 located on the right arm of chromosome IV (P61issier et al., 1995). Hydropathy plots constructed
using the algorithm of Kyte and Doolittle (Eisenberg, 1984) indicated that the aa sequence of Pho86p contains two strongly hydrophobic segments (with average hydrophobicities of 0.90 and 0.86) in its N-terminal half (Fig. 3), which may span the plasma membrane. Comparing our nt sequence data with that of Prlissier et al. (1995), which illustrated nt sequence from the BamHI2 site of P H 0 8 6 to the N C A 3 gene, we detected two polymorphisms. A T residue was inserted in Prlissier et al. (1995) D N A at a site between nt positions +806 and + 807 relative to the ATG start codon according to our data, and an 8-base segment, G G G T T C A C , at nt positions +817 to + 8 2 4 of our sequence was replaced with an A residue in Prlissier et al.'s sequence. These substitutions resulted in a difference of the aa sequences between these two nt sequence data: our P H 0 8 6 D N A encodes a :6SlLKKKGFTy276 sequence, whereas the Prlissier et al. (1995) D N A fragment encodes a I F E E K N Y sequence at the corresponding aa positions.
(b) Regulation of PH086 transcription by Pi A 1.05-kb HindlIIrHinclI 2 fragment bearing the P H 0 8 6 O R F failed to complement the pho86 mutation when it was inserted into the HindlII-NruI gap of YCp50 (pKS10; Fig. 1) and introduced into strain NS213 (pho86 pho87). This might be due to the promoter region of P H 0 8 6 in the 1.05-kb HindlII1-HinclI2 fragment being truncated at nt position - 1 2 0 relative to the A T G start codon (Fig. 2). A putative TATA box was found at nt positions - 1 0 0 to - 9 5 in the fragment. These findings suggest that there is an important nt sequence for its transcription in a region upstream from the - 1 2 0 site. We found G C A C G T G c t and aCACGTTaa sequences (lowercase letters indicate deviation from the consensus sequences, G C A C G T G G G and G C A C G T T T T , for binding of the positive regulator Pho4p; Ogawa et al., 1995b) at nt positions - 191 and - 4 9 7 (Fig. 2). Then, we investigated whether P H 0 8 6 transcription is regulated by external Pi by Northern blot hybridization with RNA samples of the wt strain, P-28-24C, cultivated in high-Pi and lowPi media (Fig. 4). We found a single hybridization band of 1.3 kb, which is consistent with the size of the P H 0 8 6 coding region. The RNA sample prepared from cells cultivated in high-Pi medium showed significant level of hybridization but slightly weaker signals than that from cells cultivated in low-Pi medium (Fig. 4). This observation is contrasted with the stringent control of the P H 0 8 4 transcription by the Pi concentration in the medium (Bun-ya et al., 1991). Thus, P H 0 8 6 is transcribed under the relaxed control of external P~. This view was also supported by an expression experiment with a fusion gene, constructed with the P H 0 8 6 promoter ligated to the P H 0 5 O R F as a reporter, in a pho5 mutant as the
44 SphZ2 -569 G C A T G C T G T A T T G C G A G T G A A ~ A G C G C A A G A C C G A G G A G A G A C A A C C C T ~ T C ~ C C C A T T A T A C A A ~ A C A C A C A C G T T A A G A G A C G C A ~ C G G C C T C G T C T G A G G A T C G T A G C C A T C A
-450 AGAGGAAGCGGAGGCGTCGAAGACCACATCGCATCGAGCGTCCACTCTCAAATATGTACCCAATAATGGAAATCCAGATGGTGGCTGTTCCACTTGCTCTTCCCAGTCCAACTGCACTGGTGCATTATCAGCAGCAGCAACAGCAGCTCC -300 CGC~CATCATCCCTGGTACGACCTCTCGCTTTCTG~GAAGCC~TCTCGACCT~CTGTTGTAGTTGACCTATAAC~GAAAATTCACACCCGCATTACCCAGCGCGCCCGCACGTG~TCTTTATGG~ACTATAATGAATAACGACCAAA HindiIi XbeI2 -150 G~CATCTTTTTTTCTCTCTCACAC`~TA~GCTTTTCATGTTTATAGATATA3AGCACATACTTTTACTAAGTTTTCTAGATTGATTACGACTCTTCCAGTTGCACATTTTTTCATAGCG~AGTACAAGAGTAGGAAGAGTGGCACA
i ATGGCGGTTC~CAAAG~d%AG~G~AGAGGGTAGA~GTCCGATAA~ATGCCCCATC~TCCCACAAGTAGATGCATCATTAGACAAACCACTTGATATTGATGCT~CTCCTACTATTTACAGTGTTAATTTGA~CCAGAATATGGT I M A V Q Q R K R K E G R K S D K N A ? S V P ~ V D A S L D K P L D I D A P P T I Y S V N L K P E Y G Pvu[Ii 151 ACAGCTGCATTG~TTTGTCCGCGGATTTCATCAGAC~G~CAGG~ATTGGCT~TAAGTATTTGTTcTT~cATC~CST~AYcC~T~TTGTTCT~ccATTGGGCTGCTTATTTACTTGACGCCAAGAATTGTCTTTCCGATAAGGAA~ 5 1 T A A L N L S A D F I R Q E Q A L A N ~ Y L F F H ? V ; L / V L T ! G L L I Y L T P R I V F P [ ~ N BamHIl FvuII2 301 ACAGGATCCGTTGCTGGATGGTTCTACCAGCT~GCTCGTATT~TAAG~GGTCGTCCTCAGCGGTTTAGTGTTCACCGCAA[TGGTGCATCTTTCTTGTTCACCCTCCTCTCACGTGTATCTGACTCGTATTTCAAGTCGAAGATCAAC 1 0 1 T G S V A G W F Y Q L A R I N K K V V L S ~ L V F ] A [ G A S F L F T L L S R V S D S Y F K S K [ N BamHi2 451 C~CTTGTTGGTTCTAAA~TGAG~AGTTTTCGGCATCAATTTGAATGATTTGGTGGC~GACATGA~c~AG~ATC~AGTGGTGAACAATACGCATATCATCGTTTATAGAGAAACGCcCATTG~ATT~ATTT~GTTAGCCCCT~C 1 5 1 Q L V G S K G E K V F G I N L N D L V A R H E T K D P V V N N T H I I V Y R E T P I A L I S L A P N
601 ATGACATT~GTACTGATGA~u~ATTTAGTCATGAGTGTTACTACTGT~GTTGTCGTCGTGTATATGTAAA~GTGGCATTATCGAAGATTTGATTGATTGGGCTATGCTACACTCCA~TATTAGAAGCAGCGGCAAATACGGGGAG 2 0 1 M T L S T D E N L V M S V T T ' J S C R R ' Y Y V ~ S S ~ [ E D L I D W A M L H S K N I R S S G K Y S Z Accll 751 ACCATG~ACTCTTGATTGATGTCTACTCATTCGATAGCACGCTTAAGGAAATTTTGAAG~AAAAGG~TTCACTTATATTCA~GCATTAGAGTCTCTGA~ATCGACTACTAGGCGGTTTGTTCGGTGTGAAGAAGGAATTATGGGGT 2 5 ! T M K L L I D V Y S F D S T L K E I L K K K G ~ T ~ [ Q S I R V S E N R L L G G L F G V K K S L W G 901 TTGC~TTCCATTTTAAAGCCGAACA~AAGGACTAGGATACTGTTATTTTTATAGCCAA~AATG~AAAA~CAGCATGGCATCTTGTTAGTTCTGTTGC~CTCGCAGGAAGTAAC~AATA~CACTATAATATTTCCATATATTTA 3 0 I L Q F H F K A E H K D * LOb1 [201 [351 L501 ~651 [801 [951
HpaI/HincIl2/AccI2 ~ATATATGTATACA~CACCTTGTTAAC~%~J~AAAAAATAAACGAACT~AGAA~CCGT~T~CAGAGTACACCTT~AAGAGAGAGAA~GAAAATCTTTGAATC~ATAGCATAATAGCGATC~CGAAACTT~TTAAGAAG~AAAATG ~CTGAAAGGAG~u%ATAT~CGGAAT~CC~TGTAT~TAATAAATAGAGAGCGGAAG~A~GAATAATAA~GGGAATTGAGGGTATCAAAATACAAGACATTCTTTTACCGAAAAGAAGAATGAC~TAG~AAC~ATTCAGC AGATCCAGAT~u%AACAGAAACTGTGCAACCATCGGAACCATCTCCGGTGAAAGAGCCATCTTCATACGCACAGCTGCCCTGAACGTTAGC~CCATCGGATGCAACTATTTTAACGTTA~GTTGGCAGCTTGGTTACTGTTGGGGTTT~G ~TC~CGACAAGTATGTTTCTCCATTAGTGGAACCAGCACCTAACACTA~TGGAGCCCAGTT~CCGACATCCGAACCAGAA~TACCCC~ATGCACCCATCTTCTGCAGATACACCGGCGTTGTTAATATAGTACTGAGCAGAAGTCTT TTTACCCTGCCATTGAT~T~GTGTCTTCATC~CGACTGA~TTGGTTGTG~TCTCCACCATCAACCACTGTGGGAATCACCATGTTTTCTGATCCTGGGTAATCCGTCCTACATAGAGCAATGGAGTCAGACTTTTTGTTAATGGC CTTAGCAGATGTTTCATCCGTAG~CATAAATCGCTGGTATCAGTGTTGGTACGGTACAAGTAACCATTTTTACAGT~A~ACCACCAACAGATTTACCATCGCTTGGTTGGTCAGAAGGCCATTGAGTCTTTGACATTC~AGGTTCACA TGCATAAGAACAGTAGTAGCCATCCTTACATTCGGACGAA~T~TTGGCATCCATGTCCATGACAGAGGCCCATCCACCAAATCCTAGCCAGTCC~GGAAACTATACCGTTTACAGAAGGG~GTCCGAACATTTGACAGTACCATCTTG
~i0[ G~TCCGCCATCACGCTTCTGTTCAGAGGTATCCTTCTCAGAT~AGGACAATGTAGTGGTAGTAGCAGCAGATTCTACA~ACTAGTAGCGGCATTGGAATCTATGTATTGAGTGACAGTGACAACCGCTGGTTTTTCATCTTTGTGATG 7251 ATC
Fig. 2. Nucleotide sequence of the PH086 gene and deduced aa sequence. The 3-kb SphI~-EcoRV fragment (the EcoRV site is on the vector moiety beside the Sau3Al/BamHt ligation site; Fig. 1) was sequenced by the routine method (Sambrook et al., 1989). The PH086 ORF starts at the + l site. The double underlines and the dashed underline in the 5'-upstream region of PH086 indicate putative binding sites ~ r the transcriptional activator, Pho4p, and a putative TATA box, respectively. The stop codon of NCA3 on the complementary strand of the indicated sequence is boxed. The nt sequence data reported have been submitted to the DDB], E MBL, and GenBank nt sequence databases under the accession No. D63817.
1.0
6)
Pi O.C
/' "
"
Y wV
'V"
v"
+
_
+
_
+
_
"
PH086
I~"
ACT1
~I~
-1.[
Residue number Fig. 3. Hydrophobicity profile of the predicted Pho86p protein. The profile was determined using the algorithm of Kyte and Doolittle wilh the normalized consensus hydrophobicity values determined by Eisenberg (1984) and a window of 21 aa. The value in parentheses is average hydrophobicity of the peak.
h o s t ( d a t a n o t shown).
We d e t e c t e d a s u b s t a n t i a l
but
l o w e r b a s a l level of P H 0 8 6 t r a n s c r i p t in the Apho4 cells grown no
in h i g h - P i m e d i u m
further
derepression
in
than
in the wt cells a n d
low-P~
medium
(Fig. 4).
M o r e o v e r , we d e t e c t e d a s o m e w h a t h i g h e r a m o u n t o f P H 0 8 6 t r a n s c r i p t in the Apho84 cells c u l t i v a t e d in h i g h Pi t h a n in the wt cells, a n d d e r e p r e s s i o n a p p e a r e d to o c c u r in low-P~ m e d i u m as in the case of the wt strain. T h e s e o b s e r v a t i o n s s u p p o r t the v i e w t h a t P H 0 8 6 is u n d e r the r e l a x e d c o n t r o l of Pi t h r o u g h the PHO r e g u l a t o r y system.
Fig. 4. Detection of the PH086 transcript by Northern blot hybridization and regulation of its expression by Pi. Samples (10 p.g) of total RNAs prepared from cells of the wt strain P-28-24C (WT), SH3319 (pho4) and SH3418 (pho84) cultivated on nutrient YPAD (nutrient high-P,: +1 or YPAD-P (nutrient low-Pc - ) medium (Bun-ya et al., 1991) were loaded into the slots. After electrophoresis (2 M HCOH/I.5% agarose), RNAs in the gel were blotted onto a nylon membrane and the filter was hybridized with a 3zp-labeled 750-bp PvuIl2-HincII2 fragment of the PH086 DNA and a 1.0-kb HindlIlXhol fragment of Sc DNA carrying ACTI (Gallwitz and Sures, 1980). The specific activity of each probe was 1.0 x 108 cpm per gg of DNA.
(c) Pho86p is a membrane protein T o d e t e r m i n e the c e l l u l a r l o c a l i z a t i o n of P h o 8 6 p , a m u l t i - c o p y p l a s m i d p Y C 2 2 5 , b e a r i n g PHO86-myc c o n struct, or v e c t o r ( Y E p l a c l 8 1 ; G i e t z a n d S u g i n o , 1988) was i n t r o d u c e d i n t o a Apho86 strain ( N B D 8 1 6 7
1A). T h e
45 Low-Pi
High-Pi
(kDa)
PHO86-myc vector PHO86-myc vector 1 S W W I S W W
214 -111 - 74--
4
m ~
¸ -~-Pho86p-myc
36
3
18-1
2
3
4
'5
6
7
8
Fig. 5. Cellular localization of Pho86p. Cells of strain NBD8167 1A (Apho86) harboring either pYC225 bearing the PHO86-myc construct (lanes 1, 2, 3, 5, 6, and 7) or vector YEplacl81 alone (lanes 4 and 8) were cultivated in synthetic high-Pi (lanes 1-4) or low-Pi (lanes 5-8) medium lacking leucine at 30°C with shaking for 16 h. Cell lysates (W; whole lysate) were prepared and fractionated into water-insoluble (I) and -soluble (S) fractions. These fractions were analyzed by Western blotting. Molecular sizes are shown at the left margin; bold number indicates the size of the Pho86p-Myc fusion protein. Methods: The PHO86-myc fusion gene was constructed as follows: The stop codon of PH086 was removed by site-directed mutagenesis using PCR (model PC-700, Astec, Fukuoka, Japan) under the following conditions: 1.5 min at 94°C, 2.5 min at 42°C, and 4 min at 72°C for 30 reaction cycles. A 0.94-kb PCR product encoding only the ORF of PH086 was cloned into TA cloning vector, pT7 Blue (Novagen, Madison, WI, USA), and the nt sequences of the inserted fragment were determined. One plasmid, pYC223, was found to have a modified PH086 DNA bearing a TAC nt sequence in place of the original stop codon, TAG. The TAC sequence was followed by a 5'-AATCGGATCCCCGGG sequence of pT7 Blue (the last 6 nt correspond to the Sinai site in MCS of pT7 Blue). A 0.89-kb HindlII1-AccI 1 fragment of PH086 (Fig. 1) was inserted into the 2.89-kb PstI-Accll (the PstI site is on MCS) gap of pYC223 with an additional 12-bp PstI-HindlII sequence derived from MCS of pUC 19 (Sambrook et al., 1989) as a linker, and the resultant plasmid was designated pYC224. A single-stranded oligodeoxynucleotide encoding the c-Myc epitope, 5'-GGGGAGCAAAAGCTCATTTCTGA AGAGGACTTGAATTGAG, was synthesized. This sense strand encodes half of the Sinai site at the 5' end, an ll-aa segment of the c-Myc epitope (Koldziej and Young, 1990), and one extra aa, followed by a TGA stop codon and recognition site for EcoRI at the 3' end. The antisense strand, 5'-AATTCTCAATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCCC, was also synthesized. These oligodeoxynucleotides were annealed, phosphorylated (Sambrook et al., 1989), and inserted into the SmaI-EcoRI gap of YEplacl81. The resultant plasmid was named pYC209. A 1.1-kb PstI-SmaI fragment containing the PH086 ORF from pYC224 was then ligated into the PstLSmaI gap of pYC209. The resultant multicopy plasmid, pYC225, carries the PHO86myc fusion gene. The cell extracts were prepared using the glass bead disruption method (Ausubel et al., 1987). Yeast cells were cultivated in 10 ml of appropriate synthetic high-Pi or low-P~ medium at 30°C for 16 h and harvested in the late logarithmic phase. After the cells were washed with 5 ml of lysis buffer (Kolodziej and Young, 1990) containing 10% glycerol/20mM HEPES (pH 7.9)/10mM EDTA/100 mM (NH4)2SO4/lmM dithiothreitol/lmM phenylmethylsulfonyl fluoride/1 Isg pepstatin A/1 pg leupeptin/2 Ixg aprotinin (all per ml), they were resuspended in the same buffer. Glass beads (GMB-60; Nippon Rikagaku Kikai, Tokyo, Japan) were then added to the cell suspension to a 1:1 (w/v) ratio, and the mixture was shaken vigorously on a Vortex
transformants were cultivated in high-Pi or low-Pi medium, whole cell extracts were prepared, and fractionated into water-soluble and insoluble fractions without addition of any detergent. The PHO86-myc construct encodes Pho86p tagged with an l l-aa segment of the c-Myc epitope at its C-terminal. The protein product of the PHO86-myc construct is functional as Pho86p, because it complemented the constitutive phenotype of rAPase in NBD8687dl (Apho86 Apho87) (data not shown). Pho86p-Myc was detected by Western analysis with monoclonal anti-c-Myc antibody. A band of Pho86p-Myc of 36 kDa, similar to the calculated value of 37 kDa including the c-Myc epitope, was clearly detected in the whole cell extract and in the insoluble fraction, while very small amounts in the soluble fraction (Fig. 5). These results suggest that Pho86p is a membrane protein or that it is associated with membrane. The same migration profiles were observed irrespective of whether these fractions were prepared from cells cultivated in high-Pi or low-Pi medium. (d) Effect of disruptions rAPase regulation
ofpho84,pho86, andpho87 on
We investigated the effect of pho86 disruption on rAPase regulation and cell viability. A 221-bp BamHI 1BamHI2 region of the PH086 DNA in pMB220 (Fig. 1) was replaced with a 2.9-kb BgllI fragment of LEU2 DNA prepared from YEpI3 (Parent et al., 1985). The resultant plasmid and the disruption allele of PH086 were designated pKS15 and pho86::LEU2, respectively (Fig. 1). A
mixer 4 times for 1 min each with chilling on ice for 1 min after each 1-min mixing (Tschopp et al., 1986). One-tenth aliquots of the cell lysates were kept as the whole cell extract fractions and the remaining fractions were centrifuged in an Eppendorf Microfuge at 104 x g for 15 min at 4°C. The resulting supernatant fraction (soluble fractions) contained water-soluble protein, and the pellet fractions contained the total-membrane fractions (Tschopp et al., 1986). The supernatant of each sample was collected and the volumes were measured. Protein concentrations in the supernatant were determined with a protein assay kit (Bio-Rad, Hercules, CA, USA) using c-Immunoglobulin G (Protein Assay Standard I; Bio-Rad) as a standard protein. The pellets were washed three times with chilled lysis buffer and resuspended in 2 × concentrated sample loading buffer (Sambrook et al., 1989) with an equal volume of corresponding supernatant fractions. This suspension was used as the insoluble fraction. A sample of the soluble fraction containing 12 ~tg protein, and ones of the insoluble fraction and the whole cell extract of the same volume as that of the soluble fraction, were loaded into slots in an 0.1% SDS-10% polyacrylamide gel, and electrophoresed. The proteins on the gel were transferred onto a nylon membrane (Immobilon PVDF, Millipore, Bedford, MA, USA). The Pho86p-Myc band was detected with a monoclonal anti-c-Myc antibody (Promega, Madison, WI, USA) at a 1/7500 dilution as the first antibody followed with an anti-mouse IgG conjugated with horseradish peroxidase (Amersham, Buckinghamshire, UK) at a 1/104 dilution. Detection of immunoreaction products was performed using the ECL Western blotting analysis system (Amersham).
46 5.4-kb HindIII1-HindIII 2 fragment containing the pho86::LEU2 allele was prepared from pKS15 and used for one-step gene disruption of the PH086 locus of a pho87::URA3 ura3 leu2 strain, SH3417, by selecting transformants on Leu test medium (Bun-ya et al., 1991). We isolated two independent Leu + transformants and found that they exhibited the rAPase + phenotype in highPi medium and arsenate resistance. This observation indicates that the Apho86 Apho87 double disruption is not lethal to the cells and confers the same phenotype as that of the original pho86-1 pho87-1 double mutation. The Pi transport system might be a complex of several proteins including Pho84p, Pho86p and Pho87p, and possibly Gtrlp (Bun-ya et al., 1992), and the complex may function under low-Pi conditions (Bun-ya et al., 1991; 1996). Alternatively, Pho84p and a complex of Pho86p and Pho87p may have similar functions but different pathways each other in the Pi uptake mechanism. If this is the case, either of a single disruption of PH084 or a double disruption of PH086 and PH087 may not be lethal, whereas triple disruption of these three genes might be lethal to the cell. To examine this possibility, we constructed such triple disruptant by crossing two disruptants: One of the Leu + transformants of SH3417 obtained in the above experiments should have the MATs pho86::LEU2 pho87::URA3 leu2 ura3 his3 genotype. This strain was crossed with SH3418 (MATa pho84::HIS3 leu2 ura3 his3; Table I). The resultant diploid was sporulated and subjected to tetrad analysis. Although the diploid hardly produced asci containing four viable spores, we isolated 50 spore-clones by dissection of 20 asci on YPAD plate. We found that six of the 50 isolates showed the His + Leu + Ura + phenotype. These six isolates showed similar phenotypes to that of the pho84 mutant, while they should have the Apho84 Apho86 Apho87 genotype. Their growth, however, was severely retarded on both high-P i and low-Pi media (data not shown), suggesting that Pi enters the cells of triple disruptant via simple diffusion mechanism.
ACKNOWLEDGEMENTS
We thank Yumi Kaburagi of our laboratory for technical assistance. This study was supported by a Grant-inAid for General Scientific Research (No. 06454079) to Y.O. from the Ministry of Education, Science, Sports and Culture of Japan, and grants to N.O. from the Nippon Life Insurance and Sumitomo Foundations.
REFERENCES Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K.: Current Protocols in Molecular Biology. John Wiley, New York, 1987.
Bun-ya, M., Nishimura, M., Harashima, S. and Oshima, Y.: The PH084 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol. Cell. Biol. 11 (1991) 3229-3238. Bun-ya, M., Harashima, S. and Oshima, Y.: Putative GTP-binding protein, Gtrl, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae. Mol. Cell. Biol. 12 { 1992) 2958-2966. Bun-ya, M., Shikata, K., Nakade, S., Yompakdee, C., Harashima, S. and Oshima, Y.: Two new genes, PH086 and PH087, involved in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr. Genet. (1996) in press. Eisenberg, D.: Three-dimensional structure of membrane and surface proteins. Annu. Rev. Biochem. 53 (1984) 595-623. Gatlwitz D., and Sures, I.: Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 77 (1980) 2546-2550. Gietz, R.D. and Sugino, A.: New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74 (1988) 527 534. Harashima S., Mizuno, T., Mabuchi, H., Yoshimitsu, S., Ramesh, R., Hasebe, M., Tanaka, A. and Oshima, Y.: Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in Saccharomyces cerevisiae. Mol. Gen. Genet. 247 (1995) 716 725. Johnston, M. and Carlson, M.: Regulation of carbon and phosphate utilization. In: Jones, E.W., Pringle, J.R. and Broach, J.R. (Eds.), The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. CSHL Press, Cold Spring Harbor, NY, 1992, pp. 193 281. Kaffman, A., Herskowitz, I., Tjian, R. and O'Shea, E.K.: Phosphorylation of the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85. Science 263 (1994) 1153-1156. Kolodziej, P.A. and Young, R.A.: Epitope tagging and protein surveillance. Methods Enzymol. 194 (1990) 508-519. Mortimer, R.K., Contopoulou C.R. and King, J.S.: Genetic and physical maps of Saccharomyces cerevisiae, edition 11. Yeast 8 (1992) 817-902. Ogawa, N. and Oshima, Y.: Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase reguIon in Saccharomyces cerevisiae. Mol. Cell. Biol. 10 (1990) 2224 2236. Ogawa, N., Noguchi, K., Yamashita, Y., Yasuhara, T., Hayashi, N., Yoshida, K. and Oshima, Y.: Promoter analysis of the PH081 gene encoding a 134 kDa protein bearing ankyrin repeats in the phosphatase regulon of Saccharomyces cerevisiae. Mol. Gen. Genet. 238 {1993 } 444 454. Ogawa, N., Noguchi, K., Sawai, H., Yamashita, Y., Yompakdee, C. and Oshima, Y.: Functional domains of Pho81p, an inhibitor of Pho85p protein kinase, in the transduction pathway of Pi signals in Saccharomyces cerevisiae. Mol. Cell. Biol. 15 (1995a} 997-1004. Ogawa, N., Saitoh, H., Miura, K., Magbanua, J.P.V., Bun-ya, M., Harashima, S. and Oshima, Y.: Structure and distribution of specific cis-elements for transcriptional regulation of PH084 in Saccharornyces cerevisiae. Mol. Gen. Genet. 249 (1995b) 406 416. Parent, S.A., Fenimore, C.M. and Bostian, K.A.: Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1 (1985) 83 138. Pdlissier P., Camougrand, N., Velours, G. and Guerin, M.: NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the ATP synthase of S. cerevisiae. Curt. Genet. 17 (1995} 409 416. Sambrook, J., Fritsch E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. CSHL Press, Cold Spring Harbor, NY, 1989. Schneider, K.R., Smith, R.L. and O'Shea, E.K.: Phosphate-regulated
47 inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science 266 (1994) 122-126. Tamai, Y., Toh-e, A. and Oshima, Y.: Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae. J. Bacteriol. 164 (1985) 964-968. Thierry, A., Fairhead, C. and Dujon, B.: The complete sequence of the 8.2 kb segment left of MAT on chromosome III reveals five ORFs, including a gene for a yeast ribokinase. Yeast 6 (1990) 521-534.
Tschopp, J.F., Emr, S.D., Field, C. and Schekman, R.: GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J. Bacteriol. 166 (1986) 313-318. Willsky, G.R., Leung, J.O., Offermann Jr., P.V., Plotnick, E.K. and Dosch, S.F.: Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae. J. Bacteriol. 164 (1985) 611-617.
41
GENE 09677
A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae (Phosphatase regulation; Pi transporter; yeast)
Chulee Yompakdee*, Masanori Bun-ya*'**, Koh Shikata, Nobuo Ogawa, Satoshi Harashima and Yasuji Oshima Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka Suita-shi, Osaka 565, Japan Received by J. Marmur: 9 November 1995; Revised/Accepted: 29 November/5 December 1995; Received at publishers: 26 January 1996
SUMMARY
The PH084 gene in Saccharomyces cerevisiae encodes the Pi transporter Pho84p. The other three genes, GTR1, PH086 and PH087, are also suggested to be involved in the Pi uptake system. We cloned and sequenced PH086 and found that it encodes a 34-kDa protein consisting of 311 amino acid residues with two strongly hydrophobic segments in its N-terminal half. Western blotting analysis of cell extracts revealed that Pho86p, tagged with c-Myc, was fractionated into a water-insoluble fraction. Disruption of PH086 did not affect cell viability even in combination with the pho84 and/or pho87 disruptions. The triple disruptants showed high levels of constitutive rAPase synthesis and arsenate resistance similar to the pho84 mutant, but showed slower cell growth than the pho84 mutant. PH086 has two putative binding sites for the transcriptional activator, Pho4p, at nucleotide positions - 1 9 1 and - 4 9 7 relative to the ATG start codon, and showed substantial levels of transcription under high-Pi conditions and more enhanced levels in low-P~ medium.
INTRODUCTION
Two systems are suggested to operate in active transport of Pi in Saccharomyces cerevisiae (Sc; Tamai et al., 1985). One has a low Km value (8.2 laM) for external Pi, and the other has high Km value (770 ~tM). PH084 has been shown to encode a major component of the Pi transporter for the low Km system (Bun-ya et al., 1991). PH084 transcription is under the stringent control of Pi concentration in the medium through the same system Correspondence to: Dr. Y. Oshima, Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan. Tel. (81-6) 879-7420; Fax (81-6) 879-7448; e-mail [email protected] * The first and the second authors contributed equally to this work. ** Present address: Faculty of Integrated Arts and Sciences, Hiroshima University, Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 724, Japan. Tel. (81-824) 24-6564. Abbreviations: aa, amino acid(s); bp, base pair(s); A, deletion; E.,
PII S0378-1119(96)00079-0
as for regulation of repressible acid phosphatase (rAPase; EC 3.1.3.2) encoded by PH05 (for review, see Johnston and Carlson, 1992). The PHO regulatory system responsive to the Pi signals consists of products of at least five genes, PH02 (=BAS2/GRFIO), PH04, PH080, PH081 and PH085. A transcriptional activator, Pho4p encoded by PH04, binds to the promoter region of the PHO structural genes. Pho2p is another transcriptional activator in the PHO genes, except PH08 encoding repressible alkaline phosphatase (EC 3.1.3.1) in vacuole, and is effective Escherichia; GTRI, gene encoding Gtrlp; Gtrlp, putative GTP-binding protein; kb, kilobase(s) or 1000 bp; MCS, multiple cloning site; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; PH04, gene encoding transcriptional activator Pho4p in the PHO reguIon; PH084, gene encoding the Pi transporter Pho84p; PH086 and PH087, genes encoding Pho86p and Pho87p, respectively, putative members of the Pi transporter complex; Pi, inorganic phosphate; rAPase, repressible acid phosphatase; Sc, Saccharomyces cerevisiae;wt, wild type; YPAD, yeast extract peptone adenine dextrose (medium); YPAD-P, YPAD medium with low Pi; : :, insertional disruption of a gene.
42 for transcription of several genes for synthesis of various aa and adenine. In high-Pi medium (containing 11.0 mM or more Pi), Pho80p (a kind of cyclin) and Pho85p (cyclin-dependent protein kinase) form a complex which inhibits Pho4p function by hyperphosphorylation of it (Kaffman et al., 1994). Then the cells are unable to enhance the transcription of PH05 and the other genes in the PHO regulon. In low-Pi conditions (0.22 mM or less P0, Pho81p inactivates the Pho80p-Pho85p complex (Schneider et al., 1994; Ogawa et al., 1995a), then Pho4p is active for transcription of the PHO genes. Transcription of PH081 is also regulated by Pi via the same PHO regulatory system (Ogawa et al., 1993). Thus, the PHO regulatory system forms a positive feedback loop. A pho84 mutation confers a rAPase + phenotype, irrespective of P~ concentration in the medium, possibly due to reduction of intracellular P~ concentration or disturbance in a Pi signal transduction mechanism. In a previous work (Bun-ya et al., 1996), we isolated a pho86 pho87 double mutant, defective in Pi uptake, by isolating mutants showing constitutive synthesis of rAPase and arsenate resistance, speculating that arsenate functions as an analog of P~ in many cellular reactions (Willsky et al., 1985) and inhibits cellular metabolism and cell growth. The single pho86 mutant, however, showed only moderate phenotypes of the double mutant and the single pho87 mutant was indistinguishable from the wt strain. Thus, PH087 plays a cooperative but auxiliary role in conjunction with PH086 in the P~-uptake system. PH087 is identical to YCR524, which was detected in the course of a Sc chromosome sequencing project (Thierry et al., 1990). The published nt sequence of PH087/ YCR524 showed that it encodes a putative 923-aa (103-kDa) membrane protein (Bun-ya et al., 1996). The aim of present studies was to characterize the PH086 gene. We found that it encodes a putative 3 l l-aa
membrane protein with two strongly hydrophobic segments in its N-terminal half. Its transcription was suggested to be under the relaxed control of Pi. Disruption of PH086 was not lethal to the cells.
RESULTS AND DISCUSSION
(a) Cloning and sequencing of PH086 To clone the PH086 gene, strain NS213 (pho86 pho87 ura3; Table I) was transformed to the Ura + phenotype with a gene library, 'CEN BANK' A (obtained from the American Type Culture Collection, Roekville, MD, USA), constructed by partial digestion of Sc DNA with Sau3AI, and ligation with YCp50 (marked with URA3; Parent et al., 1985) at the unique BamHI site. We selected three clones showing the rAPase- phenotype by staining approximately 4000 Ura ÷ colonies on high-Pi plates according to the plate assay method of rAPase as described previously (Bun-ya et al., 1991). Then, the plasmid DNAs of these Ura + rAPase- transformants were analyzed after propagation in E. coli. We found that each of these three plasmids contains an identical 4.5-kb insert (designated pMB216; Fig. 1) and confirmed that the insert is PH086 by integration of it into a homologous site in a chromosome using an integrative vector, YIp5 (Parent et al., 1985), followed by tetrad analysis of the transformant (details not shown). The restriction map (Fig. 1) of the insert apparently differed from PH084 (Bun-ya et al., 1991) and PH087 (Bun-ya et al., 1996). The essential region for the PH086 activity was narrowed down to a 2.1-kb SphIz-SpeI1 region (Fig. 1). Sequence determination of a 3-kb SphIi-EcoRV fragment (the EcoRV site is on the vector moiety beside the Sau3AI/BamH1 ligation site) revealed an ORF of 933 bp, encoding a 34-kDa protein with 311 aa residues (Fig. 2),
TABLE I Yeast strains Strain
Genotype a
Source or reference
P-28-24C NS213 SH3319 b SH3417 SH3418 NBD8167-1A c NBD8687dl c
MATa MATa MATer MATer MATa MATa MATs
Our stock Bun-ya et al. (19961 This study Bun-ya et al. (1996) Bun-ya et al. (1992) This study This study
pho3-1 pho3-1 pho86-1 pho87-1 leu2 trpl ura3 pho3-1 pho5-1 gcn4-103 pho4::LEU2 hisl-29 leu2-3, 112 ura3-52 [HIS4-1acZ, ura3-52] ura3 leu2 trpl his3 pho87::URA3 pho84::HIS3 his3-532 leu2 trpl ura3-1,2 pho3-1 leu2-3, 112 ura3-1,2 trpl-289 his3-532 ade2 canl pho86::HIS3 pho3-1 leu2-3, 112 ura3-1,2 trpl-289 his3-352 ade2 canl pho86::HIS3 pho87::URA3
a The genetic symbols used are as described by Mortimer et al. { 19921. b A derivative of strain SH3071 [PH05 ÷]Apho4 (Harashima et al., 19951 ¢ Strain NBD8687dl, a Apho86 Apho87 disruptant, was constructed from a derivative of NBW7 (Ogawa and Oshima, 1990) having the pho87::URA3 allele (Bun-ya et al., 1996) by replacement of the 1.1-kb BamHI~-Pstl segment of PH086 (Fig. 1) with a 1.2-kb BamHI-PstI HIS3 fragment of Sc prepared from YIpl (Parent et al., 19851. Strain NBD8167-1A is a haploid segregant from a diploid of the NBD8687dl x NOG1 (a derivative of NBW7t cross.
43 Pvl h S 1
E1 Sh2
Xb2 H1
B
He~JHp/A2 Pv2 A1 Spl H2 B Pv P Sa/BEV
INCA3
-,2
Plasmid
?
I
~(~) Complementation
+
pMB216 pMB217 pMB220 pKS15 pMB221 pKS8 pKS9 pKS10 pKS13
1
Chr.IV
+ J [
LEU2
I
m m
m
•
+
Fig. 1. Restriction map of the PH086 DNA and delimitation of the effective region for the PH086 function. The open and closed boxes indicate the cloned Sc DNA and the thin line the YCp50 DNA. Complementation of the DNA fragments was examined by suppression of the rAPase+ phenotype in NS213 (pho86 pho87) cells on high-Pi (YPAD) medium. The symbols + and - indicate complementation ability and inability, respectively. Abbreviations of restriction sites: A, AccI; B, BamHI; E, EcoRI; EV, EcoRV; H, HindllI; Hc, HinclI; P, PstI; Sh, SphI; Sp, SpeI; Pv, PvulI; Xb, XbaI; B/Sa, BamHl/Sau3AI; and Hc/Hp/A, HinclI/HpaI/AccI. Methods:pMB216 is the original plasmid in which the PH086 DNA was cloned on YCp50 plasmids pMB217, pMB220 and pMB221 were constructed by deleting a HindlII, SphI or XbaI fragment, respectively, from pMB216, pKS8 was constructed by eliminating a 221-bp BamHI fragment from pMB220. A 2.7-kb HindlIf~-EcoRV fragment of PH086 from pMB216 was subcloned into the HindlII-HinclI gap of pUCll9 (Sambrook et al., 1989), and the resulting plasmid was named pMB230 (not shown), pKS9 and pKS10 were constructed by inserting a 2.0-kb BamHIz-BamHl (the latter BamHI site exists on the pUC119 moiety) and a 1.2-kb HindlII1-HinclI2 fragment of PH086 from pMB230 into the BamHI site or the HindllI-NruI gap of YCp50, respectively.A 0.6-kb SpeI segment was removedfrom pMB230, and a 2.1-kb HindllIl-EcoRl (EcoRI site exists on the vector) fragment containing the SpeI deletion was inserted into the 2.7-kb HindlIIi-EcoRI gap of pMB220 to generate pKSI3. The PH086 function of the cloned fragments was examined by complementation of the rAPase÷ phenotype of strain NS213 (pho86 pho87 ura3) on high-Pi medium to the rAPase- phenotypeby introducing the plasmids (marked with URA3 bearing the fragment to be tested) into the NS213 cells.
in the 2.1-kb essential SphI2-SpeI a region. Thus, we concluded that this O R F encodes Pho86p. We also found a part of another O R F in the 3-kb sequenced fragment in tail to tail arrangement with the P H 0 8 6 ORF, but it was located outside of the 2.1-kb essential region (Fig. 1). In a search of the nt and aa sequences in the GenBank database, we found that P H 0 8 6 encodes a unique protein, and that the neighboring partial O R F is identical to N C A 3 located on the right arm of chromosome IV (P61issier et al., 1995). Hydropathy plots constructed
using the algorithm of Kyte and Doolittle (Eisenberg, 1984) indicated that the aa sequence of Pho86p contains two strongly hydrophobic segments (with average hydrophobicities of 0.90 and 0.86) in its N-terminal half (Fig. 3), which may span the plasma membrane. Comparing our nt sequence data with that of Prlissier et al. (1995), which illustrated nt sequence from the BamHI2 site of P H 0 8 6 to the N C A 3 gene, we detected two polymorphisms. A T residue was inserted in Prlissier et al. (1995) D N A at a site between nt positions +806 and + 807 relative to the ATG start codon according to our data, and an 8-base segment, G G G T T C A C , at nt positions +817 to + 8 2 4 of our sequence was replaced with an A residue in Prlissier et al.'s sequence. These substitutions resulted in a difference of the aa sequences between these two nt sequence data: our P H 0 8 6 D N A encodes a :6SlLKKKGFTy276 sequence, whereas the Prlissier et al. (1995) D N A fragment encodes a I F E E K N Y sequence at the corresponding aa positions.
(b) Regulation of PH086 transcription by Pi A 1.05-kb HindlIIrHinclI 2 fragment bearing the P H 0 8 6 O R F failed to complement the pho86 mutation when it was inserted into the HindlII-NruI gap of YCp50 (pKS10; Fig. 1) and introduced into strain NS213 (pho86 pho87). This might be due to the promoter region of P H 0 8 6 in the 1.05-kb HindlII1-HinclI2 fragment being truncated at nt position - 1 2 0 relative to the A T G start codon (Fig. 2). A putative TATA box was found at nt positions - 1 0 0 to - 9 5 in the fragment. These findings suggest that there is an important nt sequence for its transcription in a region upstream from the - 1 2 0 site. We found G C A C G T G c t and aCACGTTaa sequences (lowercase letters indicate deviation from the consensus sequences, G C A C G T G G G and G C A C G T T T T , for binding of the positive regulator Pho4p; Ogawa et al., 1995b) at nt positions - 191 and - 4 9 7 (Fig. 2). Then, we investigated whether P H 0 8 6 transcription is regulated by external Pi by Northern blot hybridization with RNA samples of the wt strain, P-28-24C, cultivated in high-Pi and lowPi media (Fig. 4). We found a single hybridization band of 1.3 kb, which is consistent with the size of the P H 0 8 6 coding region. The RNA sample prepared from cells cultivated in high-Pi medium showed significant level of hybridization but slightly weaker signals than that from cells cultivated in low-Pi medium (Fig. 4). This observation is contrasted with the stringent control of the P H 0 8 4 transcription by the Pi concentration in the medium (Bun-ya et al., 1991). Thus, P H 0 8 6 is transcribed under the relaxed control of external P~. This view was also supported by an expression experiment with a fusion gene, constructed with the P H 0 8 6 promoter ligated to the P H 0 5 O R F as a reporter, in a pho5 mutant as the
44 SphZ2 -569 G C A T G C T G T A T T G C G A G T G A A ~ A G C G C A A G A C C G A G G A G A G A C A A C C C T ~ T C ~ C C C A T T A T A C A A ~ A C A C A C A C G T T A A G A G A C G C A ~ C G G C C T C G T C T G A G G A T C G T A G C C A T C A
-450 AGAGGAAGCGGAGGCGTCGAAGACCACATCGCATCGAGCGTCCACTCTCAAATATGTACCCAATAATGGAAATCCAGATGGTGGCTGTTCCACTTGCTCTTCCCAGTCCAACTGCACTGGTGCATTATCAGCAGCAGCAACAGCAGCTCC -300 CGC~CATCATCCCTGGTACGACCTCTCGCTTTCTG~GAAGCC~TCTCGACCT~CTGTTGTAGTTGACCTATAAC~GAAAATTCACACCCGCATTACCCAGCGCGCCCGCACGTG~TCTTTATGG~ACTATAATGAATAACGACCAAA HindiIi XbeI2 -150 G~CATCTTTTTTTCTCTCTCACAC`~TA~GCTTTTCATGTTTATAGATATA3AGCACATACTTTTACTAAGTTTTCTAGATTGATTACGACTCTTCCAGTTGCACATTTTTTCATAGCG~AGTACAAGAGTAGGAAGAGTGGCACA
i ATGGCGGTTC~CAAAG~d%AG~G~AGAGGGTAGA~GTCCGATAA~ATGCCCCATC~TCCCACAAGTAGATGCATCATTAGACAAACCACTTGATATTGATGCT~CTCCTACTATTTACAGTGTTAATTTGA~CCAGAATATGGT I M A V Q Q R K R K E G R K S D K N A ? S V P ~ V D A S L D K P L D I D A P P T I Y S V N L K P E Y G Pvu[Ii 151 ACAGCTGCATTG~TTTGTCCGCGGATTTCATCAGAC~G~CAGG~ATTGGCT~TAAGTATTTGTTcTT~cATC~CST~AYcC~T~TTGTTCT~ccATTGGGCTGCTTATTTACTTGACGCCAAGAATTGTCTTTCCGATAAGGAA~ 5 1 T A A L N L S A D F I R Q E Q A L A N ~ Y L F F H ? V ; L / V L T ! G L L I Y L T P R I V F P [ ~ N BamHIl FvuII2 301 ACAGGATCCGTTGCTGGATGGTTCTACCAGCT~GCTCGTATT~TAAG~GGTCGTCCTCAGCGGTTTAGTGTTCACCGCAA[TGGTGCATCTTTCTTGTTCACCCTCCTCTCACGTGTATCTGACTCGTATTTCAAGTCGAAGATCAAC 1 0 1 T G S V A G W F Y Q L A R I N K K V V L S ~ L V F ] A [ G A S F L F T L L S R V S D S Y F K S K [ N BamHi2 451 C~CTTGTTGGTTCTAAA~TGAG~AGTTTTCGGCATCAATTTGAATGATTTGGTGGC~GACATGA~c~AG~ATC~AGTGGTGAACAATACGCATATCATCGTTTATAGAGAAACGCcCATTG~ATT~ATTT~GTTAGCCCCT~C 1 5 1 Q L V G S K G E K V F G I N L N D L V A R H E T K D P V V N N T H I I V Y R E T P I A L I S L A P N
601 ATGACATT~GTACTGATGA~u~ATTTAGTCATGAGTGTTACTACTGT~GTTGTCGTCGTGTATATGTAAA~GTGGCATTATCGAAGATTTGATTGATTGGGCTATGCTACACTCCA~TATTAGAAGCAGCGGCAAATACGGGGAG 2 0 1 M T L S T D E N L V M S V T T ' J S C R R ' Y Y V ~ S S ~ [ E D L I D W A M L H S K N I R S S G K Y S Z Accll 751 ACCATG~ACTCTTGATTGATGTCTACTCATTCGATAGCACGCTTAAGGAAATTTTGAAG~AAAAGG~TTCACTTATATTCA~GCATTAGAGTCTCTGA~ATCGACTACTAGGCGGTTTGTTCGGTGTGAAGAAGGAATTATGGGGT 2 5 ! T M K L L I D V Y S F D S T L K E I L K K K G ~ T ~ [ Q S I R V S E N R L L G G L F G V K K S L W G 901 TTGC~TTCCATTTTAAAGCCGAACA~AAGGACTAGGATACTGTTATTTTTATAGCCAA~AATG~AAAA~CAGCATGGCATCTTGTTAGTTCTGTTGC~CTCGCAGGAAGTAAC~AATA~CACTATAATATTTCCATATATTTA 3 0 I L Q F H F K A E H K D * LOb1 [201 [351 L501 ~651 [801 [951
HpaI/HincIl2/AccI2 ~ATATATGTATACA~CACCTTGTTAAC~%~J~AAAAAATAAACGAACT~AGAA~CCGT~T~CAGAGTACACCTT~AAGAGAGAGAA~GAAAATCTTTGAATC~ATAGCATAATAGCGATC~CGAAACTT~TTAAGAAG~AAAATG ~CTGAAAGGAG~u%ATAT~CGGAAT~CC~TGTAT~TAATAAATAGAGAGCGGAAG~A~GAATAATAA~GGGAATTGAGGGTATCAAAATACAAGACATTCTTTTACCGAAAAGAAGAATGAC~TAG~AAC~ATTCAGC AGATCCAGAT~u%AACAGAAACTGTGCAACCATCGGAACCATCTCCGGTGAAAGAGCCATCTTCATACGCACAGCTGCCCTGAACGTTAGC~CCATCGGATGCAACTATTTTAACGTTA~GTTGGCAGCTTGGTTACTGTTGGGGTTT~G ~TC~CGACAAGTATGTTTCTCCATTAGTGGAACCAGCACCTAACACTA~TGGAGCCCAGTT~CCGACATCCGAACCAGAA~TACCCC~ATGCACCCATCTTCTGCAGATACACCGGCGTTGTTAATATAGTACTGAGCAGAAGTCTT TTTACCCTGCCATTGAT~T~GTGTCTTCATC~CGACTGA~TTGGTTGTG~TCTCCACCATCAACCACTGTGGGAATCACCATGTTTTCTGATCCTGGGTAATCCGTCCTACATAGAGCAATGGAGTCAGACTTTTTGTTAATGGC CTTAGCAGATGTTTCATCCGTAG~CATAAATCGCTGGTATCAGTGTTGGTACGGTACAAGTAACCATTTTTACAGT~A~ACCACCAACAGATTTACCATCGCTTGGTTGGTCAGAAGGCCATTGAGTCTTTGACATTC~AGGTTCACA TGCATAAGAACAGTAGTAGCCATCCTTACATTCGGACGAA~T~TTGGCATCCATGTCCATGACAGAGGCCCATCCACCAAATCCTAGCCAGTCC~GGAAACTATACCGTTTACAGAAGGG~GTCCGAACATTTGACAGTACCATCTTG
~i0[ G~TCCGCCATCACGCTTCTGTTCAGAGGTATCCTTCTCAGAT~AGGACAATGTAGTGGTAGTAGCAGCAGATTCTACA~ACTAGTAGCGGCATTGGAATCTATGTATTGAGTGACAGTGACAACCGCTGGTTTTTCATCTTTGTGATG 7251 ATC
Fig. 2. Nucleotide sequence of the PH086 gene and deduced aa sequence. The 3-kb SphI~-EcoRV fragment (the EcoRV site is on the vector moiety beside the Sau3Al/BamHt ligation site; Fig. 1) was sequenced by the routine method (Sambrook et al., 1989). The PH086 ORF starts at the + l site. The double underlines and the dashed underline in the 5'-upstream region of PH086 indicate putative binding sites ~ r the transcriptional activator, Pho4p, and a putative TATA box, respectively. The stop codon of NCA3 on the complementary strand of the indicated sequence is boxed. The nt sequence data reported have been submitted to the DDB], E MBL, and GenBank nt sequence databases under the accession No. D63817.
1.0
6)
Pi O.C
/' "
"
Y wV
'V"
v"
+
_
+
_
+
_
"
PH086
I~"
ACT1
~I~
-1.[
Residue number Fig. 3. Hydrophobicity profile of the predicted Pho86p protein. The profile was determined using the algorithm of Kyte and Doolittle wilh the normalized consensus hydrophobicity values determined by Eisenberg (1984) and a window of 21 aa. The value in parentheses is average hydrophobicity of the peak.
h o s t ( d a t a n o t shown).
We d e t e c t e d a s u b s t a n t i a l
but
l o w e r b a s a l level of P H 0 8 6 t r a n s c r i p t in the Apho4 cells grown no
in h i g h - P i m e d i u m
further
derepression
in
than
in the wt cells a n d
low-P~
medium
(Fig. 4).
M o r e o v e r , we d e t e c t e d a s o m e w h a t h i g h e r a m o u n t o f P H 0 8 6 t r a n s c r i p t in the Apho84 cells c u l t i v a t e d in h i g h Pi t h a n in the wt cells, a n d d e r e p r e s s i o n a p p e a r e d to o c c u r in low-P~ m e d i u m as in the case of the wt strain. T h e s e o b s e r v a t i o n s s u p p o r t the v i e w t h a t P H 0 8 6 is u n d e r the r e l a x e d c o n t r o l of Pi t h r o u g h the PHO r e g u l a t o r y system.
Fig. 4. Detection of the PH086 transcript by Northern blot hybridization and regulation of its expression by Pi. Samples (10 p.g) of total RNAs prepared from cells of the wt strain P-28-24C (WT), SH3319 (pho4) and SH3418 (pho84) cultivated on nutrient YPAD (nutrient high-P,: +1 or YPAD-P (nutrient low-Pc - ) medium (Bun-ya et al., 1991) were loaded into the slots. After electrophoresis (2 M HCOH/I.5% agarose), RNAs in the gel were blotted onto a nylon membrane and the filter was hybridized with a 3zp-labeled 750-bp PvuIl2-HincII2 fragment of the PH086 DNA and a 1.0-kb HindlIlXhol fragment of Sc DNA carrying ACTI (Gallwitz and Sures, 1980). The specific activity of each probe was 1.0 x 108 cpm per gg of DNA.
(c) Pho86p is a membrane protein T o d e t e r m i n e the c e l l u l a r l o c a l i z a t i o n of P h o 8 6 p , a m u l t i - c o p y p l a s m i d p Y C 2 2 5 , b e a r i n g PHO86-myc c o n struct, or v e c t o r ( Y E p l a c l 8 1 ; G i e t z a n d S u g i n o , 1988) was i n t r o d u c e d i n t o a Apho86 strain ( N B D 8 1 6 7
1A). T h e
45 Low-Pi
High-Pi
(kDa)
PHO86-myc vector PHO86-myc vector 1 S W W I S W W
214 -111 - 74--
4
m ~
¸ -~-Pho86p-myc
36
3
18-1
2
3
4
'5
6
7
8
Fig. 5. Cellular localization of Pho86p. Cells of strain NBD8167 1A (Apho86) harboring either pYC225 bearing the PHO86-myc construct (lanes 1, 2, 3, 5, 6, and 7) or vector YEplacl81 alone (lanes 4 and 8) were cultivated in synthetic high-Pi (lanes 1-4) or low-Pi (lanes 5-8) medium lacking leucine at 30°C with shaking for 16 h. Cell lysates (W; whole lysate) were prepared and fractionated into water-insoluble (I) and -soluble (S) fractions. These fractions were analyzed by Western blotting. Molecular sizes are shown at the left margin; bold number indicates the size of the Pho86p-Myc fusion protein. Methods: The PHO86-myc fusion gene was constructed as follows: The stop codon of PH086 was removed by site-directed mutagenesis using PCR (model PC-700, Astec, Fukuoka, Japan) under the following conditions: 1.5 min at 94°C, 2.5 min at 42°C, and 4 min at 72°C for 30 reaction cycles. A 0.94-kb PCR product encoding only the ORF of PH086 was cloned into TA cloning vector, pT7 Blue (Novagen, Madison, WI, USA), and the nt sequences of the inserted fragment were determined. One plasmid, pYC223, was found to have a modified PH086 DNA bearing a TAC nt sequence in place of the original stop codon, TAG. The TAC sequence was followed by a 5'-AATCGGATCCCCGGG sequence of pT7 Blue (the last 6 nt correspond to the Sinai site in MCS of pT7 Blue). A 0.89-kb HindlII1-AccI 1 fragment of PH086 (Fig. 1) was inserted into the 2.89-kb PstI-Accll (the PstI site is on MCS) gap of pYC223 with an additional 12-bp PstI-HindlII sequence derived from MCS of pUC 19 (Sambrook et al., 1989) as a linker, and the resultant plasmid was designated pYC224. A single-stranded oligodeoxynucleotide encoding the c-Myc epitope, 5'-GGGGAGCAAAAGCTCATTTCTGA AGAGGACTTGAATTGAG, was synthesized. This sense strand encodes half of the Sinai site at the 5' end, an ll-aa segment of the c-Myc epitope (Koldziej and Young, 1990), and one extra aa, followed by a TGA stop codon and recognition site for EcoRI at the 3' end. The antisense strand, 5'-AATTCTCAATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCCC, was also synthesized. These oligodeoxynucleotides were annealed, phosphorylated (Sambrook et al., 1989), and inserted into the SmaI-EcoRI gap of YEplacl81. The resultant plasmid was named pYC209. A 1.1-kb PstI-SmaI fragment containing the PH086 ORF from pYC224 was then ligated into the PstLSmaI gap of pYC209. The resultant multicopy plasmid, pYC225, carries the PHO86myc fusion gene. The cell extracts were prepared using the glass bead disruption method (Ausubel et al., 1987). Yeast cells were cultivated in 10 ml of appropriate synthetic high-Pi or low-P~ medium at 30°C for 16 h and harvested in the late logarithmic phase. After the cells were washed with 5 ml of lysis buffer (Kolodziej and Young, 1990) containing 10% glycerol/20mM HEPES (pH 7.9)/10mM EDTA/100 mM (NH4)2SO4/lmM dithiothreitol/lmM phenylmethylsulfonyl fluoride/1 Isg pepstatin A/1 pg leupeptin/2 Ixg aprotinin (all per ml), they were resuspended in the same buffer. Glass beads (GMB-60; Nippon Rikagaku Kikai, Tokyo, Japan) were then added to the cell suspension to a 1:1 (w/v) ratio, and the mixture was shaken vigorously on a Vortex
transformants were cultivated in high-Pi or low-Pi medium, whole cell extracts were prepared, and fractionated into water-soluble and insoluble fractions without addition of any detergent. The PHO86-myc construct encodes Pho86p tagged with an l l-aa segment of the c-Myc epitope at its C-terminal. The protein product of the PHO86-myc construct is functional as Pho86p, because it complemented the constitutive phenotype of rAPase in NBD8687dl (Apho86 Apho87) (data not shown). Pho86p-Myc was detected by Western analysis with monoclonal anti-c-Myc antibody. A band of Pho86p-Myc of 36 kDa, similar to the calculated value of 37 kDa including the c-Myc epitope, was clearly detected in the whole cell extract and in the insoluble fraction, while very small amounts in the soluble fraction (Fig. 5). These results suggest that Pho86p is a membrane protein or that it is associated with membrane. The same migration profiles were observed irrespective of whether these fractions were prepared from cells cultivated in high-Pi or low-Pi medium. (d) Effect of disruptions rAPase regulation
ofpho84,pho86, andpho87 on
We investigated the effect of pho86 disruption on rAPase regulation and cell viability. A 221-bp BamHI 1BamHI2 region of the PH086 DNA in pMB220 (Fig. 1) was replaced with a 2.9-kb BgllI fragment of LEU2 DNA prepared from YEpI3 (Parent et al., 1985). The resultant plasmid and the disruption allele of PH086 were designated pKS15 and pho86::LEU2, respectively (Fig. 1). A
mixer 4 times for 1 min each with chilling on ice for 1 min after each 1-min mixing (Tschopp et al., 1986). One-tenth aliquots of the cell lysates were kept as the whole cell extract fractions and the remaining fractions were centrifuged in an Eppendorf Microfuge at 104 x g for 15 min at 4°C. The resulting supernatant fraction (soluble fractions) contained water-soluble protein, and the pellet fractions contained the total-membrane fractions (Tschopp et al., 1986). The supernatant of each sample was collected and the volumes were measured. Protein concentrations in the supernatant were determined with a protein assay kit (Bio-Rad, Hercules, CA, USA) using c-Immunoglobulin G (Protein Assay Standard I; Bio-Rad) as a standard protein. The pellets were washed three times with chilled lysis buffer and resuspended in 2 × concentrated sample loading buffer (Sambrook et al., 1989) with an equal volume of corresponding supernatant fractions. This suspension was used as the insoluble fraction. A sample of the soluble fraction containing 12 ~tg protein, and ones of the insoluble fraction and the whole cell extract of the same volume as that of the soluble fraction, were loaded into slots in an 0.1% SDS-10% polyacrylamide gel, and electrophoresed. The proteins on the gel were transferred onto a nylon membrane (Immobilon PVDF, Millipore, Bedford, MA, USA). The Pho86p-Myc band was detected with a monoclonal anti-c-Myc antibody (Promega, Madison, WI, USA) at a 1/7500 dilution as the first antibody followed with an anti-mouse IgG conjugated with horseradish peroxidase (Amersham, Buckinghamshire, UK) at a 1/104 dilution. Detection of immunoreaction products was performed using the ECL Western blotting analysis system (Amersham).
46 5.4-kb HindIII1-HindIII 2 fragment containing the pho86::LEU2 allele was prepared from pKS15 and used for one-step gene disruption of the PH086 locus of a pho87::URA3 ura3 leu2 strain, SH3417, by selecting transformants on Leu test medium (Bun-ya et al., 1991). We isolated two independent Leu + transformants and found that they exhibited the rAPase + phenotype in highPi medium and arsenate resistance. This observation indicates that the Apho86 Apho87 double disruption is not lethal to the cells and confers the same phenotype as that of the original pho86-1 pho87-1 double mutation. The Pi transport system might be a complex of several proteins including Pho84p, Pho86p and Pho87p, and possibly Gtrlp (Bun-ya et al., 1992), and the complex may function under low-Pi conditions (Bun-ya et al., 1991; 1996). Alternatively, Pho84p and a complex of Pho86p and Pho87p may have similar functions but different pathways each other in the Pi uptake mechanism. If this is the case, either of a single disruption of PH084 or a double disruption of PH086 and PH087 may not be lethal, whereas triple disruption of these three genes might be lethal to the cell. To examine this possibility, we constructed such triple disruptant by crossing two disruptants: One of the Leu + transformants of SH3417 obtained in the above experiments should have the MATs pho86::LEU2 pho87::URA3 leu2 ura3 his3 genotype. This strain was crossed with SH3418 (MATa pho84::HIS3 leu2 ura3 his3; Table I). The resultant diploid was sporulated and subjected to tetrad analysis. Although the diploid hardly produced asci containing four viable spores, we isolated 50 spore-clones by dissection of 20 asci on YPAD plate. We found that six of the 50 isolates showed the His + Leu + Ura + phenotype. These six isolates showed similar phenotypes to that of the pho84 mutant, while they should have the Apho84 Apho86 Apho87 genotype. Their growth, however, was severely retarded on both high-P i and low-Pi media (data not shown), suggesting that Pi enters the cells of triple disruptant via simple diffusion mechanism.
ACKNOWLEDGEMENTS
We thank Yumi Kaburagi of our laboratory for technical assistance. This study was supported by a Grant-inAid for General Scientific Research (No. 06454079) to Y.O. from the Ministry of Education, Science, Sports and Culture of Japan, and grants to N.O. from the Nippon Life Insurance and Sumitomo Foundations.
REFERENCES Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K.: Current Protocols in Molecular Biology. John Wiley, New York, 1987.
Bun-ya, M., Nishimura, M., Harashima, S. and Oshima, Y.: The PH084 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol. Cell. Biol. 11 (1991) 3229-3238. Bun-ya, M., Harashima, S. and Oshima, Y.: Putative GTP-binding protein, Gtrl, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae. Mol. Cell. Biol. 12 { 1992) 2958-2966. Bun-ya, M., Shikata, K., Nakade, S., Yompakdee, C., Harashima, S. and Oshima, Y.: Two new genes, PH086 and PH087, involved in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr. Genet. (1996) in press. Eisenberg, D.: Three-dimensional structure of membrane and surface proteins. Annu. Rev. Biochem. 53 (1984) 595-623. Gatlwitz D., and Sures, I.: Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 77 (1980) 2546-2550. Gietz, R.D. and Sugino, A.: New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74 (1988) 527 534. Harashima S., Mizuno, T., Mabuchi, H., Yoshimitsu, S., Ramesh, R., Hasebe, M., Tanaka, A. and Oshima, Y.: Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in Saccharomyces cerevisiae. Mol. Gen. Genet. 247 (1995) 716 725. Johnston, M. and Carlson, M.: Regulation of carbon and phosphate utilization. In: Jones, E.W., Pringle, J.R. and Broach, J.R. (Eds.), The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. CSHL Press, Cold Spring Harbor, NY, 1992, pp. 193 281. Kaffman, A., Herskowitz, I., Tjian, R. and O'Shea, E.K.: Phosphorylation of the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85. Science 263 (1994) 1153-1156. Kolodziej, P.A. and Young, R.A.: Epitope tagging and protein surveillance. Methods Enzymol. 194 (1990) 508-519. Mortimer, R.K., Contopoulou C.R. and King, J.S.: Genetic and physical maps of Saccharomyces cerevisiae, edition 11. Yeast 8 (1992) 817-902. Ogawa, N. and Oshima, Y.: Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase reguIon in Saccharomyces cerevisiae. Mol. Cell. Biol. 10 (1990) 2224 2236. Ogawa, N., Noguchi, K., Yamashita, Y., Yasuhara, T., Hayashi, N., Yoshida, K. and Oshima, Y.: Promoter analysis of the PH081 gene encoding a 134 kDa protein bearing ankyrin repeats in the phosphatase regulon of Saccharomyces cerevisiae. Mol. Gen. Genet. 238 {1993 } 444 454. Ogawa, N., Noguchi, K., Sawai, H., Yamashita, Y., Yompakdee, C. and Oshima, Y.: Functional domains of Pho81p, an inhibitor of Pho85p protein kinase, in the transduction pathway of Pi signals in Saccharomyces cerevisiae. Mol. Cell. Biol. 15 (1995a} 997-1004. Ogawa, N., Saitoh, H., Miura, K., Magbanua, J.P.V., Bun-ya, M., Harashima, S. and Oshima, Y.: Structure and distribution of specific cis-elements for transcriptional regulation of PH084 in Saccharornyces cerevisiae. Mol. Gen. Genet. 249 (1995b) 406 416. Parent, S.A., Fenimore, C.M. and Bostian, K.A.: Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1 (1985) 83 138. Pdlissier P., Camougrand, N., Velours, G. and Guerin, M.: NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the ATP synthase of S. cerevisiae. Curt. Genet. 17 (1995} 409 416. Sambrook, J., Fritsch E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. CSHL Press, Cold Spring Harbor, NY, 1989. Schneider, K.R., Smith, R.L. and O'Shea, E.K.: Phosphate-regulated
47 inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science 266 (1994) 122-126. Tamai, Y., Toh-e, A. and Oshima, Y.: Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae. J. Bacteriol. 164 (1985) 964-968. Thierry, A., Fairhead, C. and Dujon, B.: The complete sequence of the 8.2 kb segment left of MAT on chromosome III reveals five ORFs, including a gene for a yeast ribokinase. Yeast 6 (1990) 521-534.
Tschopp, J.F., Emr, S.D., Field, C. and Schekman, R.: GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J. Bacteriol. 166 (1986) 313-318. Willsky, G.R., Leung, J.O., Offermann Jr., P.V., Plotnick, E.K. and Dosch, S.F.: Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae. J. Bacteriol. 164 (1985) 611-617.