Mol Gen Genet (1991) 229:30%315 002689259100310H © Springer-Verlag 1991

DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains* Rolf Stucka 1, Sylvie Dequin 2, Jean-Michel Salmon 2, and Carlos Ganeedo 3 1 Institut ffir Physiologische Chemic, Biochemie und Zellbiologie der Universit/it, W-8000 M/inchen 2, Federal Republic of Germany 2 Laboratoire de Microbiologie et Technologic des Fermentations, INRA-IPV, F-34060 Montpellier, France 3 Instituto de Investigaciones Biom6dicas C.S.I.C., Facultad de Medicina U.A.M., E-28029 Madrid, Spain Received March 5, 1991

Summary. A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes. Key words: Pyruvate carboxylase - Chromosome II Chromosome VII - Saccharomyces- P YC1, PYC2

be useful for study of the interaction between glycolysis and other pathways leading to oxaloacetate. In the yeast Saccharomyces cerevisiae, a mutant without significant pyruvate carboxylase activity was reported by Wills and Melham (1985). However it was shown later that technical problems in the enzymatic assay rendered the conclusion of lack of activity unwarranted (Lira et al. 1987). Therefore we decided to try to obtain mutants lacking pyruvate carboxylase activity. During our work we discovered that there are two genes coding for pyruvate carboxylase in S. cerevisiae. One of them corresponds to the gene reported by Morris et al. (1987) the sequence and domain structure of which have been elucidated by Lira et al. (1988). We report here on the isolation of the second pyruvate carboxylase gene and the chromosomal localization of each of the genes. The sequences of both genes are quite homologous in the coding region but present significant differences in the non-coding part. We also show by disruption experiments that expression of either gene alone provides enough activity to allow growth in the absence of exogenous aspartate, while disruption of both genes results in aspartate auxotrophy.

Materials and methods Introduction Synthesis of oxaloacetate from pyruvate is an important reaction both for gluconeogenesis and for replenishment of the tricarboxylic acid cycle. In eukaryotic organisms this carboxylation reaction is catalysed by pyruvate carboxylase (for reviews see Utter etal. 1975; Scrutton 1978). Mutants defective in pyruvate carboxylase would * A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990) Offprint requests to: C. Gancedo

Strains, vectors and culture conditions. S. cerevisiae W303-1A MATa ade2-1 trpl-1 leu2-3, 112, his3-11,15 ura3 canl-lO0 or the isogenic strain W303-1B MAT~ ade2-1 trpl-1 leu2-3,112 his 3-1115 ura3 canl-lO0 were used. Yeasts were grown in 1% yeast extract, 2% bactopeptone, 2% glucose (YPD) or in a defined minimal medium containing 0.17% yeast nitrogen base (Difco) and 0.5% ammonium sulfate with the appopriate supplements as required and 2% glucose as carbon source. For some experiments ammonium sulfate was replaced by aspartate or glutamate at the same molar concentration. Liquid cultures were grown in a rotary shaker. Growth temperature was 30 ° C. Strains harbouring plasmids were grown on media lacking the requirement cor-

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responding to the selective marker on the plasmid. Crosses and sporulation of yeast strains were performed by conventional techniques. E. coli strain DH5 (Sambrook et al. 1989) was used for plasmid propagation and isolation. For sequencing, E. coli strain JM103 (Pharmacia, Freiburg, FRG) was used in combination with M13 derivatives (Vieira and Messing 1982). Bacteria were grown with shaking at 37 ° C in LB medium supplemented with 50 gg/ml ampicillin if required. Constructions were done in plasmids pUC18 (Yanisch-Perron et al. 1985), YEp352 or YEp351 (Hill et al. 1986b). DNA manipulations. DNA manipulations were done by standard methods (Sambrook et al. 1989). Probes were labelled as described by Feinberg and Vogelstein (1983). Restriction fragments from the different plasmids were subcloned after isolation from low melting point agarose gels. Sequencing was performed using the dideoxy chaintermination method (Sanger et al. 1977). Oligonucleotide synthesis was performed on a BioSearch 8600 DNA synthesizer. Yeast transformation was carried out as described by Ito et al. (1983) or Burgers et al. (1987). Construction of plasmids carrying the PYC1 or PYC2 genes and disruption of these genes in the S. cerevisiae genome. Using the oligonucleotide 5'-TAAACTTCTGGATCTGGGCC-3' corresponding to positions 25362555 of a published sequence for the pyruvate carboxylase gene (Lira et al. 1988) we screened a yeast genomic library (kindly provided by M. Crouzet, Universit6 Bordeaux I, France). A positive clone, pSD1, showed a restriction map consistent with the reported sequence. A 6.5 kb SaII-EcoRI fragment was obtained by partial digestion and subcloned in vector pRS316 (Sikorski and Hieter 1989). The 6.5 kb SaII-XbaI fragment from this

construction was inserted into YEp351 to produce pCG50 (Fig. 1). To disrupt the PYC1 gene, the internal 1155bp PstI fragment (Fig. 1) was subcloned into pUC18 (Vieira and Messing 1982) and a 3 kb BglII fragment of YEpl3 (Broach et al. 1979), containing the yeast LEU2 gene, was inserted into the unique BglII site of the P YC1 fragment. The 4.1 kb PstI fragment of this construct was used to replace the homologous segment of the resident copy of P YC1 in strain W303-1A (Fig. 1). To disrupt the PYC2 gene, a 6 kb SalI fragment of cosmid clone c411 was subcloned into YEp352 to yield plasmid pRSI (Fig. 1). The 4144 bp XhoI-SmaI fragment of pRS1 carrying the PYC2 gene was subcloned into YEp352 (plasmid pRS2) and a 1.2 kb BglII fragment containing the yeast URA3 gene from pFL34 (kindly donated by F. Lacroute, Paris, France) was inserted into the unique BamHI site of PYC2. The 3.4 kb HpaI fragment from this construct was used to replace the homologous segment of the resident copy of PYC2 in strain W303-1B (Fig. 1). One-step gene replacement was done according to Rothstein (1985) and Leu + or Ura + colonies were selected. Chromosomal disruptions were checked by Southern hybridization. The multicopy plasmids pCG50 and pRS2 were used for overexpression of the PYC1 and PYC2 genes. For chromosomal localization of PYC1 and PYC2, transverse alternating field electrophoresis of chromosomal DNA was performed as in Vezinhet et al. (1990). The D N A was transferred to nitrocellulose membranes and probed with an oligonucleotide specific for the coding region of the pyruvate carboxylase gene (see below). The autoradiogram was exposed for 1 week. Extracts and assay of enzyme activities. Extracts were prepared by shaking 20 mg yeast with 200 mg glass beads (0.5 mm in diameter) in 0.2 ml of 0.1 M MES, 0.1 M KC1, 10 mM MgC12, pH 6.5 for 4 min. The beads were removed by centrifugation. For determination of kinetic parameters, pyruvate carboxylase was partially purified as follows: cells were shaken with glass beads (1 g yeast/10 g glass beads) in 10 ml of 0.1 M TRIS-HC1, 10 mM MgC12, 5 mM EDTA, 0.1 mM dithioerythritol, 1 mM phenylmethylsulfonyl fluoride, pH 7.2. The beads were removed by filtration and the filtrate was centrifuged at 6000 × g for 20 rain. The supernatant was centrifuged for 2 h at 100000 x g, precipitated with 0.04% protamine sulfate and then centrifuged at 8000 x g for 20 min. The supernatant was precipitated with solid ammonium sulfate at 50% saturation and the precipitate was extracted with ammonium sulfate solutions of different concentrations as described by Young et al. (1969). The fraction extracted at 35% saturation was dialysed against extraction buffer and used for enzymatic assays. Pyruvate carboxylase was assayed in a coupled assay with citrate synthase by following the formation of CoA from acetyl CoA as described by Martin and Denton (1970). The dependence of activity on ATP and its inhibition by avidin were routinely confirmed. Assay temperature was 30 ° C; protein was measured according to Lowry et al. (1951).

309 Results

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Identification o f a second yeast pyruvate carboxylase gene in S. cerevisiae

We tried to isolate mutants lacking pyruvate carboxylase by mutagenesis of yeast and selection for cells able to grow on aspartate-supplemented media but unable to do so on unsupplemented ones. The rationale behind this approach was that, in aspartate-supplemented media, aspartate transaminase would be able to supply the mutant with the oxaloacetate produced normally in the wild type by pyruvate carboxylase. However in spite of multiple attempts the desired mutant could not be obtained. At least two explanations may be offered for this result: either a lack of the enzyme is lethal for the cells or more than one functional gene coding for the enzyme exists in yeast. To obtain the mutant and to distinguish between the two aforementioned possibilities we decided to isolate the pyruvate carboxylase gene, to disrupt it and to replace the resident genomic copy by the disrupted one. For this purpose, the oligonucleotide 5'-TAAACTTCTGGATCTGGGCC-3', corresponding to positions 25362555 of the published sequence of the gene coding for pyruvate carboxylase (Lim et al. 1988), was synthesized and used to probe a preparation of yeast chromosomes separated by transverse alternating field electrophoresis. In four different strains tested two bands hybridizing with the probe were always observed, one corresponding th chromosome VII and another one corresponding to chromosome II (Fig. 2). In a cosmid collection isolated by chromosomal walking of the whole of chromosome II (R. Stucka, P. Nelb6ck, C. Schwarzlose and H. Feldmann, in preparation), the region that hybridized with the probe was contained in a 6 kb SalI fragment of cosmid c411. This cosmid overlaps with cosmid cyril18, which has been mapped to a specific region of chromosome II carrying a tRNA ~Iu3 gene, a Tyl-ll8 element and the DUR1,2 loci (Eigel et al. 1981; Genbauffe etal. 1984; Stucka et al. 1986). In cosmid c411 a sequence was found that presented homology with the one reported for the pyruvate carboxylase-encoding gene (Lira et al. 1988) but showed a number of differences (Fig. 3). These results strongly suggested the existence of more than one gene coding for pyruvate carboxylase. To test this, we screened a total yeast cosmid library (Hauber et al. 1988) with the 2.1 kb BamHI-XhoI fragment from plasmid pRS1 (Fig. 1) situated in the region of homology between the two sequences. Five cosmids gave a positive signal; three of them overlapped with c411 while the other two showed a different restriction map consistent with that of the previously reported gene coding for pyruvate carboxylase (Lim et al. 1988). We sequenced a 3 kb B a m H I fragment from one of these cosmids and found that the sequence was identical with the one reported by Lim et al. (1988). Therefore it appears that in S. eerevisiae two genes exist which are able to code for pyruvate carboxylase. We designate the gene located on chromosome VII corresponding to the one first re-

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Fig. 2. Localizationof PYC1 and PYC2 on the yeastchromosomes. A preparation of yeast chromosomal DNA from four different strains was separated by transverse alternatingfieldelectrophoresis (see the Materials and methods). S. cerevisiae strains used were as follows: lane i, AB972 MATe SUC2 mal reel gal2 CUPI; lane 2, DC04 MATe adel leu2 cir°; lane 3, A364A MATe adel ade2 ural his7 lys2 tyrl gall; lane 4, 4NN281 MAT~ trpl-d his3-d,200 ura3-52 lys2-80! ade2-1 gal. On the figure Roman numerals indicate the chromosomenumber ported as P Y C I and the one on chromosome II as PYC2.

Characteristics o f the sequence of PYC2

The open reading frame corresponding to P Y C 2 (Fig. 3) codes for a protein of 1185 amino acids, which is seven amino acids longer than P Y C 1 , due to an additional serine residue at the amino-terminus and six other amino acids at the carboxyl-end. A comparison of the sequence of P Y C 2 with that of P Y C I showed an overall homology at the amino acid level of 92%. The degree of homology on the nucleotide sequence level is ca. 85%. This difference is mainly due to 297 conservative substitutions in wobble positions. A high degree of conservation is found in the regions corresponding to the binding sites for ATP and pyruvate (96% homology at the amino acid level) while divergence is observed in the biotinyl carrier region with a homology of only 67%. The positions of the sequences coding for these domains are indicated in the legend to Fig. 3. A calculation of the codon bias index (Bennetzen and Hall 1982) showed a value of 0.55 for P Y C 1 and of 0.57 for P Y C 2 corresponding to that of moderately expressed genes (Sharp et al. 1986) with a bias in certain amino acids of P Y C 2 towards the usage found in highly expressed genes. In both genes, canonical forms of the yeast TATA box (Harbury and Struhl 1989) were found: in P Y C 1 the sequence TATATA is found twice at positions - 1 1 8 and - 1 1 0 while P Y C 2 shows no evident TATA box in analogous posi-

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TTCGTAGAAAAA TGATTCGTGCATTAATCGAGTTCAGAATTAC~TGTCAAGACCAACATT CCCTTCCTATTGACTCTTTi GACCAATCCAGTATTTAT T ~ T A ~ T A C T ~

226l ,u63

F I D D T P 0 t F 0 H V S S 0 X R A Q K t t H Y t A D t A V N G 5 5 I K G 0 I G 50~ CTTTTATTGACGACACCCCACAACTGTTCC~TGGTATCATCACAAAACAGAGCGC~CTGTTACACTATTTGGCAGACTTGGCAGTTAACGGT TCTTCT A T T ~ T ~ T T G 3360 IIIIIIIIIIIIIIIIIIIIllllfllllllllllll Illlllllllllll II IIIIIlll IIIll fl I II Ill IIIIII II 11111 Illll Illllllllllll CTTTTATTGACGACACCCCACAACTGT TCCA.~TGGTTTC~TCACA/~CAGG~C~CTT TTACATTACCTCGCCGACGT~GA~T~ TCATCTATC~T~TTG V

Fig. 3 (legend see opposite page)

D

2~8]. .$03

L p K L ~ S N P S V P H L H D A 0 G N V I N V T K S A P P S G W R Q V L L E K G 5~ GCTTGCCAAAACTA~TCAAATCCAAGTGT CCCCCATTTGCACGATGCTCA~TGTCATCAACGT TACAA.AGTCTGCACCACCA TCCGGATGGAGACAAGTGCTAC TGGAAAA~ N S 0 IIIIIIIIII •••••••••••••••••••••••••••••••••••••••••••••••••••••••••|•••••••••••••••••••••••••••••• IIIllllllll IIIIIII 2501 GCTTGCCAAAATT~U~AATCAAAT CCAAGTGTCCCCCATI' TGCACGATGCTCAGGGC~TGTCATCAACGA fI CAAAGTCTGCACCACCATCCCC-~ATGGAGGCAAG TGCTACT A ~

5q3 p C E F A K 0 V R 0 F N G T L L n D T T W R D A H 0 S L L A T R V R T H D L A T 58~ GACCATGTGAATTTGCCAAGC~GT CAGACAGTTCAAIGGTACTCTACTGATC~CACCACC T~CGCTCATCAA TCTCT A C T T ~ G T ~ C C~C~TTT~CTA 360~ III IIIIIllllll t r i l l 1111111 I I I I I l l l l l II i i I i i i i f l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l 2621 GGCCAC~TGAA1"TTGCCAC~CAN~TTAGAC/~'i3"CAATGGTACTTTATTGATGGACACCACCT ~ C G C T ~ T ~ T C ] CTACT T G ~ G T~C C~C~T~T~ A

583

R

I A P T T A H A L A G A F A L E C W G G A T F D V A ~ R F L H E D P W E R L R K CAArCGCTCCAACAACCGCACATGCCCTT~C TTTCCC , TTTAC~TGTTCGGGTGGTGCGACATfCGACGTTGCAAT~TTCTT~T~T , CCAT~CGTCT~ IIIIIlllllllllllllllllllllllllll IIIIIIII Illlllllllllllllllll IIIIIIII IIIII111111111 IIIIIIIIIII1111111 IIIII 11111

CAATCC,CTCCAACAACCGCACATGCCCTTGCAC..~3TGCTT TCGCCTTAC~AA'IGTTC,K]CGTC~3TGCCACATTCC.~ATG1-f , ~T~T/TTT~T~T

C~T~CGT~ 0

62q 3720 27ul 623

L R S L V P N I P F 0 H L L R G A T G V A Y S S L P D X A I D H F V K 0 A K D N 66~ AATTAAGATCTCTGGTC-CCTAATATTCCATTCCA~TGI~ATTGCGTGGTGCCACT~TGTGGCT TACTCTTCATTACCTGACAAT G C T A T T G A C ~ T T ~ G T ~ C ~ T A 38q0

IIIIIIIIit1111111111111111111111 I I I I I I I l l l l l l l l l l l l l

Illlllllllll

II111111 I I I I I I I I I I I I I I I I I I I I I I I

IIIIIllllllll~llll

2861 663

A~TTA`~C~AT~T~TGGTGC~TAATATTCCATT~CA~TGTTATTGCGTGGTG~CAATGGTGTGGCTTATTCTTCATT~CT~TGCTA~C~T~CGT~~TA X

G V D I F R V F D A L N D L E O L K V G V N A V K K A G G V V E A I" V C Y ~ G D 70~ ATGGTGTTGATATATTTAGAGTCTTTGATC, CC/TGA,ATGATTTAGAACAATTAAAAGTTGGTGTGAATGCTGTCAAGAAGGCC~T~TG~GTC~CTAC TGTTTGTTACTCT~TG 39f:~ I iiiiiiiiiiiiiiiiiiiiiiiiiiiiii lllll II llllllll II II IIIII IIIIIII llllllll IIIIIIIIIII IIIII IIIIIIIIII IIIIII ATAGTGTTGATATATT%~.~GTCTFiG ' ATGCCTTAAATGACTTGGAACAATT~ CGGTGTAGATGCTGT~GGTGGTGTTGTAC~CTGT~GTTTCT C T ~ 2981 703 S F ~ L 0 P G K K Y N L D Y Y L E V V E K I V O ~ G T H I L G I K D tl A G T t~ K P A 7uU ACATGCFTCAGC~TAAC~TAC~CTTAGATTACTACC TAGAAGTTGTTGAA~TAGTTCA~TGGGTACACATAT CT T G G G T A ~ T A T ~ T A C T A T ~ C C ~ ~080 Illlllllllllll IIlIIIIIIII II I I l l l l l l I III III IIIIIIlll II I l l l l l l l II I l l l l l Illllll II I l l l l l l l l l l l l l IIIII II

310X Zq3

ATATGC~TCAGCCAC~AC~TTGGATTACTACTTGGAAATTGCTC~TTGTC~kAATC~CTCATATC~TGGGTATCAAAGATATGSCAC~TAC~T~ I

A

A A K L L I G S L R R Y P D L P I H V H S H D S A S T R V A S n T A C A L A G 78~ ~200 CCGCTGCCAAATTATTAATTGGCTCCCTAAC~CCAC~TATCCGGAT TTACCAATTCATGTTCACAGTCATGACTCC~TACTCGTGTTCC ' GTCTATGACTGCATGT~CCTAG~ IIIIIIIII II I IIIII II I II I II II III I IIIII IIIIIIIIII III II 1! Ill IIIIIIIIIIIII II IIIIIIII IIIII II II

CACW~TGCCA.AACTACTGATTGGATCITT~CTAAGTACCCTGATCTC cr.~a,~TACATGTTCACACTCACGATTCTGCAGGTACTCGTGTTGCATCAAT~CT ~ G T G T ~ T C T ~ A K

T

G

32"21 F83

A D V V D V A I N S /i S G L T S Q P S I N A L L A S L E G N I D T G I N V E H V 82q GTGCTGATGTTGTCGATGTAGCTATCAA TTCAATG~ ' CGGGCTTAACTTCCCAACCATCAATTAATGCAC TGTTGGCTTCATT A F - ~ T ~ T T ~ T A C T ~ T T ~ C G T T ~ T G q320 II I I I I I II I I I l l II I l l l l IIIIIlll II I I I I I I I I IIIIIIIIIII IIIII IIIIIIIIIIIIIIIIIIIIIII Illll IIIII 111111111111111 GCGCCGATGTCGTTGATGTTGCCATCAACTCAA TGTCTGGTTTAACTTCACAACCATC~TCAA , TGCTCTGTTGGCTTCATT ~ T ~ T A T T ~ C A C T ~ T A T T ~ T T ~ T G 33~i ~23 R E L D A Y R A E tl R L L Y P C F E A D L K G P D P E V Y g H E I P G G 0 L T N 86~ TCCGTGAATTAGATGCATATACC, C~CGAAATC,~.~CTGTTGTATCCTTGTTT CGAC~CCGACTTC~CCAC~TCCAG,~TTTACCAACATGAAAT CCCAGGTGGT~ T T ~ C TA ~ Illllll IIIIIIlllll I l l l II I I I I I I I l l l If IIIIIllllllll I/llllllllllllllllllllllllll,I IIIIIIlllllllllllllllllllllll N61 TCCGTC.P~CTAGATGCATATTGGGCJLGAGATGAGATTGTTATACTCTTGTTTC~TGACTTGAAGC~]-CCCAC~ATCCAG,a.a,~TTTATCJkACATGAAATCCCA~T~T ~ T T ~ 863 W S L L F 0 A 0 O L G L G E 0 W A E T K R A Y R E A N Y L L G D | V K V T P T S K V 9~ ACTTGTTATTCCAAGCTC.AACAACTGGGTCTTGGTGAACAATGGGCTGAAACTAAAAGAC-CTTACAGAGA.a.GC~TTAC CTACT~TA~GTT~GTTACCCC~CTTC T ~ ~56~ Illllll II I I I I I IIIIII IIIIIIIIII Illllllllll IIII Illlllllllllllllllllllllll tl I I I I I I I I I I I I IIIIIIIIIIIIIIIII IIII

3581 903 V G D L A 0 F tl V S N K L T S D D I R R L A N S L D F P D S V ~I D F F E G L l G _~ T~GTCG~TGAT.ITAGCTCAATTCATGGTTTCTAACAAACTGA~TT~GACGATATTAGACGTTTAGCT~CTTT~CTTTC~T~TC~GTTAT~CT~TTT~TTT~TG~f~0

ACTTGTTGTTTC.AACW~CCAACAATTGGGTCTTGGAGAACAAT~C.~SGCCCAAACAAA,a.aE.~kGCTTA~GCCAATTATTTATTGGGTGATATfGT~TTACCCCAACTTC~ Q

II llllll l II

IIII lllll II II III I IIIIIIII III I lllll

I llllllllllllll

II IIIIIIIIIIIIIIIII

II II lllll IIIII I

T~G~GGTGA~CTC.~ITTA~GG~CAATAAATTAACT~CGA~GATG~GAGA~GCCTC~TAA~T~TTTGSATT~CC~TGA~C~GT~A~GGAT~~T~T~G K V

3701 ~$

~ P Y G G F P E P L R S D V L R N K R R K L T C R P G L E L E P F D L E K I

GTCAACCATACGGTC.,CW~ TTCC~ C C A TTAAC~ATCTGATGTATT~CAAC~ACCkAC~;kAGTTGACGTGCCGTC ~ T I

TAGAATTAC~AACC A ~

TCTC ~

R E

TTAnG ~800

llllllll llllllllllllllllllll IIIII II II II II llllllllllllllllllll II IIIIIIII I III llll lllllllllllllllllllllllll GCCAACCATATGGTGGGTICC~CCATTTAGATCAGACGTTTTAAGGAA~~TTGACTTG'I CGTCCAG3CCTGGAACT~CA~T~TCTC~TTA~

3821 9~$

D L 0 N R F G D I D E C D V A S N N t i Y P R V Y E D F 0 K l R E T Y G D L S V L 102~ AAGACTT~CAC.~TTCGG TC~ATATTC,~TGAATC~CGATGTTGCTTCTA~CAATATGTATC C ~ CTATC~,~.~C.C~T TTCC A A , ~ A C . - A T ~ T A C ~ T~ l TTAT ~ C ~920 IIIII11111[I

IIIII

IIIIII

IIIIIII

Illll

IIIIlllll

I II

II111

IIlll

II

IIIIIlll

lllllllllll

IIIIIlll

II

Illllllllll

II

~CTI'GCAGAATAGATTTGGTGATGTTGATGAGTGCGACGTTGCTTCTTATAAr..ATGTACCCAAGAGTFI'AT~CTTC~T~CGTATGGT~I TTATCTGTAT 39~1 V Y 1023 P T K N F L A P A E P D E E I

E V T I

IIIII

IIIII

E O G K T L

I

I

K L 0 A V G D L N K K T G

TACCAACCAAaA~.TTT~CT~C`CA~CAGCAGAACC~GA~T~CA~CATCGAA~TAAGACTTTGATTATC~A.~ATTG~TGTT~T~CT~T~ I II

II

I

I 11|

III

IIII

I~11111

I1[111111~1111

II

I I111~111

I II

IIIII

IIIII

II

II

II

II

II

106~

TG ~0~0 I

~06~ 1063 0 R E V Y F. E L N G E L R K I R V A D K S 0 N I 0 S V A K P K A D V H D T H 0 1 110~ $160 ~TGTATT TTGAATTGAACGGTC#k~TT~vE ' ,A,~C.4~T CAGAGI~GCAGA~TCAC.~CATACAATC T G I - I G C T ~ C ~ I ~T GTC~C~TACI~ C ~ IIIIIIIIII II I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I llllllIl I II I I I I I I I I I I I II II II I II III I II I GT ~ T T T A C T T T G A T T T G A A I G G I G ~ T G A C - A A ~ A ~ . T T C G T G T T G C T ~ + ~ ~ C T G ~ A C T ~ T C ~ T ~ T ~ T C ~ T T A ~~181 110~ E D /i R K V E T T $ tl P L H G A P /i A G V I I. E V K V H K G S L V K K G E S I A V L S A tl K ~ E tl V V S S P 11~ TCGGTGCACCAATGGCTGGTGTTATCATAG, A,~TT ' AAAGTACATAAAG"~ T C T T T G G T ~ G A A TCGAI~C ' ,CTG~TT ~ T ~ T ~ T ~ T ~ T T G TCTCTT~C $280 [llllllfllllll Illll III Iflllllllll Illlllll II I I IIIIIIIII 11 I I II II II II I I I I I l l l l l l l l l l l l l II I I I I I I I TTGGT~CC,AATGGCAGGTGTCATTGTTGAAGTTAA~AGTTCATAAAC~-~TCA~TAATAAAC4~GGGCCAA~TGTAGCCGTA1-TAA~I~T~ T~TTATATCT TCTC ~01 v I o p v I I 1.1~3 A D G 0 V K D V F I R D G E S V D A S D L L V V L E E E T L P P S P K K V I F T 11Bq CAGCAC~TGGTCA,aGTTAAAGATGI~ 'T ' [CATTAGGGATGGTGAA~TGTTGACGCA TC~T TTGTTGGTTGTCCTAC~CCCTACCC C ~ T C C C ~ T ~ T T T T TA WOO III Illll lllllllllll II If I IIIIIIIlll I I I I I I I II I l l l l If 111 I I I I I I I I II I I I I I II CAT~CC~TGGACAAGTTA~GTG1TTGT~T~TGA~(~TGAAAATGTGGACT~TT~TGATTTATTAG1-TCTATTAGAAGACCAAG~TC~TGTT~CT~T~CC~TAGT ~21 S E V $ N S L D 0 v P v E T K A 117~ R 11~ CTCGTTAAT~ATATTTTAT~ACATCTGa,~TACTAGC T(~TACTATATA'~GGCGTATAT'~TTATCTAGI~ATGTTCCAT~TATATTT ~ T ~ T ~ G T ~ T ~ C T T TC~ ~$20 I II I II I I I III I I I III I I I I I I II I II , I II I I 11 I I I ! ! TCTCATTTATAATGTATA~TATACCCGAATCTTATTTATTTACCTTTCCTATTTTTTGACGACCAGTAAATACTAATA~T~TT~TT~T~T~TT ~5~1 ATGA~ATAC~TGCTAACTG"i'GTI'TCTTCC~TAATGCTTT~ACTTACCAT~TCTCCATTCi'CCATTTTCT~CTTC~AGT~TGT~T~ TAT~C~T~TACG TT~CTACTCTA~ 56~0 I IIII I II I I I I II I I I I I I II I III I II I TAACGCATCCAAT TAACGTGTCCTTTITTCATEATTAATTTATCTACTATTTCGA~ ' TTAAATTCCATATACAATAAATCCTAGATACATTCCCGAAAGT~TCTTT T A ~ T C T T u661 TTAATATCG'J'ACGGC,Ar i'l~ £GATC.GACT~TAGGTTTTC'i'TCTT,AGACC~TTC~GC~CGC~ 57~ I I I I I ! I I III I II II III CCTTGAGCTGCTAGCAGTGGGCrTAGTCCACCTGTTAGTTACTCTTGGTAIACCACTAGGTCTT ~725 T G C ~ / I R S

| ~1GTCTCCACTAGAGACTGACGAAC4k~ATTGA,AGTTGTAATCGAACAAGGTA.a, AAC,GCTAATTATCAAGCT A C A G G C T G T ~ T ~ T ~ C C G S

L

T

V

Fig, 3. Sequence of PJ{C2 and comparison with that of P Y C 1 . Only the coding strand is represented. The 5'non-coding regions of P YC2 and P YC1 are shown for up to 1850 and 875 nucleotides, respectively, upstream of the first ATG. The upper row corresponds to the sequence of P Y C 2 and the lower one to that of P Y C 1 . Amino acids in P Y C 1 which differ from those o f P Y C 2 are shown.

Domains for ATP, pyruvate and biotinyl carrier were located from the data o f Lim et al. (1988) for P Y C 1 and occupy the following positions: ATP domain A, from 157 to 333; ATP domain B, from 353 to 488; pyruvate domain from 558 to 913; biotinyl carrier from •089 to 1178

312 Table 1. Sequences of the 5'regions of P YC1 and P YC2 with putative binding sites for regulatory factors" Binding site or factor

Consensus sequence

Sequence in gene b

Gene in which sequence occurs

Reference

ABFI/GF1 GRF2/REB1 CT block GCN4 CDEI TREe

RTCRYNNNNNACG c YNNYYACCCG CTTCC TGACTC RTCACRTG GGTCATGACC

GTCACTAACGACG -492 CCCTTACCCG -476 CTTCCTGCGGAAG -403 TGACTC a -278 GTCACGTG -488 GGTCATGACC - 330 GGTCATGCTC - 146

PYC2 PYC2 PYC2 PYC2 PYC1 PYC1 PYC1

Buchmann et al. (1988) Chasman et al. (1990) Chambers et al. (1990) Hill et al. (1986a) Mellor et al. (1990) Glass et al. (1988)

A review on regulatory DNA-binding proteins and binding sequences in yeast has appeared recently (Verdier 1990) b Numbers refer to the position of the first base represented, taking as + 1 the first ATG of the ORF ° Y represents a pyrimidine, R a purine d Sequence present in opposite orientation with respect to the mRNA start site e TRE, thyroid response element

Table 2. Growth and pyruvate carboxylase activity of strains carrying disruptions of the genes PYC1 and PYC2 Saccharomyces cerevisiae strain

W 303-1A I-3 II-2 22A ° 22B 22C 22D

Relevant genotype

PYC1 PYC2 pycl::LEU2 PYC2 P YC1 pye2:: URA3 pyel : :LEU2 PYC2 pycl" :LEU2 pye2: : URA3 PYC1 PYC2 PYClpyc2::URA3

Generation time (h) a

Pyruvate carboylase b mU/mg protein

i

ii

4 4 5 3.7 ng d

2.5 2.5 3 3.7 2.2

26 18 20 20 nd e

4 3.6

2.6 2.8

28 11

a Cells were grown in minimal medium with (i) ammonium sulfate or (ii) aspartate as sole nitrogen source b Activities were assayed in extracts of cells grown on YPD harvested during the exponential phase of growth c Strains 22A, 22B, 22C and 22D are the products of a tetratype tetrad derived from a cross between I-3 and II-2 d ng, no growth end, not detectable

tions. In P YC2 the incomplete T A T A T sequence is found at position - 6 5 and the sequence T A T T T A is located at - 2 9 0 from the first A T G . Table 1 shows a series of motifs of potential regulatory significance in the non-coding regions of P Y C 1 and P Y C 2 . A B F I binding sites have been found in several glycolytic promoters like E N 0 2 and T D H 3 (Rhode et al. 1989; D o r s m a n et al. 1990) and P G K o r P Y K ( C h a m b e r s et al. 1990) and appear to act synergistically with other weak transcriptional activators to achieve a high level of transcription (Nishizawa et al. 1989). Comparison of the A B F I sequence element of the P Y C 2 gene with that of a number of yeast genes revealed a perfect match with a sequence in the UAS (upstream activating sequence) element of the E N 0 2 p r o m o t e r (Cohen et al. 1986). The c o m m o n m o t i f reads 5 ' - G T C A C T A A C -

G A C G T G - 3 ' . The G R F 2 sequence element has been described as having little activity by itself but is a strong activator of transcription when placed at less than 20 bp from other activating elements (Chasman et al. 1990). In the p r o m o t e r of P Y C 2 the sequences ABF1 and G R F 2 are separated by three bases only. This suggests that ABF1 and G R F 2 could act synergistically. The expression of P Y C 2 m a y be further modulated by the CT block which is found also in some glycolytic genes (Chambers et al. 1990). In the P YC2 upstream region a C T T C C sequence is separated by 3 bp f r o m its inverted sequence G G A A G . We have also found a possible G C N 4 binding site; to our knowledge this has not previously been found in genes coding for enzymes related to carbohydrate metabolism. The p r o m o t e r of P Y C 1 contains the C D E I sequence whose function has not been clearly established although it appears in front of several promoters like G A L 2 or T R P I and was shown to be a target binding site for the C P F I protein (Mellor et al. 1990). It was surprising to find that this p r o m o t e r contains the consensus sequence of the thyroid response element.

Disruption and overexpression o f the yeast pyruvate carboxylase genes

To study the function of the D N A regions that appear to encode pyruvate carboxylase, we disrupted the P Y C 1 and P Y C 2 genes by insertion of the L E U 2 and the U R A 3 gene respectively (Fig. 1) and substituted the resident genomic copies by the disrupted ones. Correct integration of the disruption genes was checked by Southern blot analysis (results not shown). As mentioned before, it was expected that a lack of pyruvate carboxylase activity would result in a requirement for aspartate. However the introduction of either the p y c i : : L E U 2 or the p y c 2 : : U R A 3 disruption into the yeast genome did not produce a requirement for aspartate or other marked changes in the parameters assayed (Table 2). A cross between strains carrying p y c i : : L E U 2 and pyc2: : U R A 3

313 A

B

C

Table 3. Kinetic parameters of pyruvate carboxylase isoenzymes from S. eerevisiae

D

kb 23.1

_

Parametera

9.4_ ~

Enzymecoded by PYC1

PYC2

Reportedvalues (Ruiz-Amil et al. 1966)

0.45

0.31

0.80

0.19

0.20

0.24

0.31

0.17

no data

_ PYC 2

65-

Kmfor pyruvate (mM) Kmfor ATP (mM) Kinact at 37° C (rain- 1)

4.3_

2.3_ 2.0-

a The parameters were determinedusingpartiallypurifiedpreparations obtained from strains carryingonly one functionalPYC gene ~

_ PYC 1

Fig. 4. Southern blot analysis of chromosomai D N A from strains with disruptions in genes PYC1, PYC2 or both. Chromosomal D N A from the spores of a tetratype tetrad derived from a cross between strains I-3 (pyci : :LEU2) and II-2 (pyc2: : URA3) was digested with PstI, electrophoresed, transferred to nitrocellulose and probed with 32p-labelled 1. I kb PstI fragm'ent internal to the P YC1 gene. Relevant genotypes are as follows: lane A, pyci::LEU2 PYC2; lane B, pycl: :LEU2 pyc2:: URA3; lane C, PYCI PYC2; lane D, PYC1 pyc2::URA3. Pyruvate carboxylase activities of these strains are shown in Table 2. Figures indicate the size of D N A in kb produced the expected 2:2 segregation pattern of the selective markers among the progeny. One segregant of a tetratype tetrad failed to grow on glucose minimal medium unless aspartate was added. Glutamate could not replace aspartate as sole nitrogen source for this mutant when glucose was used as carbon source. A Southern analysis of the DNA of the segregants of this tetrad is shown in Fig. 4. Spore 22B shows bands resulting from a disruption of both P Y C I and PYC2 and this strain shows the aspartate-requiring phenotype. Spore 22C exhibited bands corresponding to intact genes while spores 22A and 22D showed disruptions of PYC1 and PYC2 respectively. An analysis of growth and enzymatic activity of the segregants of this tetrad is presented in Table 2. Strain 22D carrying the disruption pyc2:: URA3 presented a somewhat lower enzymatic activity than 22A, which carries the p y c l : : L E U 2 disruption, and is also less active than the parental strain carrying the pyc2::URA3 disruption. Since both parentals are isogenic we have no explanation for this result. The activity found in cells carrying the two functional alleles is approximately equal to the sum of the activities found in strains with individual disruptions. These results support our conclusion that S. cerevisiae has two functional pyruvate carboxylase genes which can fulfil the same role. The lack of growth in the absence of aspartate when both genes are disrupted shows that in normal conditions there is no physiological bypass for the carboxylation of pyruvate. When a multicopy plasmid carrying the P YC1 or

the PYC2 gene was introduced into wild-type yeast a ca. twofold increase in the specific activity of pyruvate carboxylase was observed. However, when both genes carried in multicopy plasmids were simultaneously introduced into the yeast no additional increase in enzymatic activity could be measured.

Characteristics of the enzymes encoded by PYC1 and PYC2

Pyruvate carboxylase was partially purified from strains carrying only one functional allele. No significant differences in behaviour were observed between the two preparations. Affinity constants for ATP and pyruvate did not differ significantly and agreed fairly well with those reported in the literature for "pyruvate carboxylase" (Table 3). Also the sensitivity to aspartate was similar for both enzymes. However the enzyme coded by PYC1 lost up to 100% activity after overnight storage at 4° C even in the presence of ammonium sulfate.

Discussion

The results presented show unequivocally that in S. cerevisiae there are two genes able to code for pyruvate carboxylase. It is clear from the growth behaviour of yeasts carrying individual disruptions in each of the genes that each of them is able to perform the same enzymatic function. The occurence of two genes poses the question of their physiological significance; in yeast many duplicated functional genes exist and several explanations may be offered for their existence. In some cases these genes encode isoenzymes with different cellular localizations (Kim et al. 1986; Rickey and Lewin 1986), while in others their expression is differentially regulated by growth conditions (Mc Alister and Holland 1982, 1985) and, moreover, one of the products may have a regulatory role in addition to the catalytic one (Entian and Fr6hlich 1984). In the case of pyruvate carboxylase no indication of differences in subcellular localization of the two enzymes has been found: all reports of subcellular localization of the enzyme, based on activity measurements that would not differentiate between

314 the products of the two genes, clearly describe it as cytoplasmic (Haarasilta and Taskinen 1977; van U r k et al. 1989). A cytosolic localization was also demonstrated by immunocytochemical labelling (Lim etal. 1987), which due to the similarities of the two proteins would probably detect both of them. Moreover neither the sequence of P Y C 1 nor that of P Y C 2 appears to possess a mitochondrial targeting signal. We do not presently know how the transcription of P YC1 or P YC2 is regulated. The differences in the upstream regions could indicate differential expression of these genes. In this respect it m a y be of significance that the sequence element 5 ' - G T C A C T A A C G A C G T G - 3 ' which is found in the UAS of the glucose-inducible E N 0 2 gene and appears necessary for induction by glucose (Cohen et al. 1986) is also present in the P Y C 2 upstream region. Since it has been reported that the levels of enzymatic activity remain at a fairly constant level under a variety of growth conditions (Ruiz-Amil et al. 1965; Cazzulo et al. 1968; Haarasilta and Oura 1975) it m a y be speculated that P Y C 1 is transcribed in the absence of glucose. In relation to the presence in P Y C 1 of consensus sequences of the thyroid responsive element, it m a y be of interest to mention the recent result of Privalsky et al. (1990) showing that the v-erbA oncogene, an altered copy of a cellular gene for a thyroid h o r m o n e receptor, is functional as an activator of transcription in yeast. Taking into account the high degree of h o m o l o g y between the coding regions of the P Y C 1 and P Y C 2 genes, it is likely that they are the product of a duplication event with the resulting genes being distributed on different chromosomes, as also appears to be the case for the phosphofructokinase genes (Heinisch et al. 1989). The abrupt divergence of the two peptide sequences at the carboxy-terminus can be related to a peculiarity in the nucleotide sequence of the P Y C 2 gene: a symmetric sequence C C C T A C C C C C A T C C C m a r k s the breakpoint of the homologous region and m a y be a consequence of the duplication event. Both enzymes show similar characteristics in all tests used as might be expected from the high h o m o l o g y of the gene sequences. It is worth mentioning that in spite of this similarity the stability of the protein encoded by P YC1 is much less than that of the enzyme encoded by PYC2. We cannot at present offer any explanation for this difference. A noteworthy feature is that only a moderate increase in activity was observed when multicopy plasmids carrying both P YC1 and P YC2 genes were introduced into wild-type yeast. Several explanations m a y be offered for this result. Either the enzyme that attaches the biotin to the apo-carboxylase or biotin itself m a y be present in limiting amounts in the cell so that even if apoenzyme is produced in large quantities no increase in enzyme activity beyond a given threshold can occur. Alternatively the expression of the genes or the translation into active product could be controlled by the a m o u n t of the corresponding m R N A , as found for some other genes (Moore etal. 1990; H o h m a n n and Cederberg 1990). The fact that two genes for pyruvate carboxylase

are found in several strains of different origin explains previous failures to obtain mutants lacking pyruvate carboxylase using classical mutagenesis methods. The resuits of the disruption experiments clearly show that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. Acknowledgements. We thank H. Feldmann and P. Barre for continuous interest and support. The critical reading of the manuscript by Juana M. Gancedo and the expert technical assistance of Christa Schwarzlose are greatly appreciated. We thank M. Crouzet for the gift of the yeast genomic library. This work was partially supported by grants to H. Feldmann from the Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (SFB 190, Miinchen), to Juana M. Sempere from the DGICYT (PB87-0294), to C. Gancedo from the EEC (BAP 0389.E (JR)), and from the program of "Acciones Integradas Hispano - Francesas" to the Montpellier and Madrid laboratories. R.S. was the recipient of a short-term FEBS Fellowship.

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