Yeast 14: 963–967 (1998)

Isolation and Nucleotide Sequence of the GeneEncoding Phosphoenolpyruvate Carboxykinase fromKluyveromyces lactis

HIROKO K. KITAMOTO1*, SADAHIRO OHMOMO2 AND YUZURU IIMURA3

1National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305, Japan2National Institute of Animal Industry, Kukisakimachi Ikenodai 2, Inashiki-gun, Ibaraki 305, Japan3Department of Applied Chemistry and Biotechnology, Yamanashi University, Takeda 4-3-11, Koufu,Yamanashi 400, Japan

Received 26 November 1997; accepted 12 February 1998

The KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK; ATP-dependent) was cloned from theKluyveromyces lactis genome using a PCR amplicon from Saccharomyces cerevisiae PCK1 gene as a probe. A DNAfragment of about 4·8 kb containing KlPCK1 complemented PEPCK activity of the mutant of S. cerevisiae defectivein PEPCK. The KlPCK1 gene has an open reading frame of 1629 bp (543 amino acids). The KlPCK1 nucleotidesequence and deduced amino acid sequence showed 76% and 84% homologies to those of S. cerevisiae PCK1,respectively. Multiple alignment of ATP-dependent PEPCK genes shows highly conserved regions. The nucleotidesequence of KlPCK1 has been submitted to the DDBJ/GenBank/EMBL data bank with Accession Number U88575.? 1998 John Wiley & Sons, Ltd.

— gluconeogenesis; PEPCK; Kluyveromyces lactis

*Correspondence to: H. K. Kitamoto, National Institute ofAgrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki305, Japan. Tel.: (+81) 298 38 7452; fax: (+81) 298 38 7408;

INTRODUCTION

Growth of yeasts on non-carbohydrate carbonsources requires gluconeogenesis, the reversalof glycolysis. Two enzymes are characteristicin gluconeogenesis, fructose-1,6-bisphosphatase(E.C. 3.1.3.11) and phosphoenolpyruvate car-boxykinase (E.C. 4.1.1.49; PEPCK). A mutant ofSaccharomyces cerevisiae lacking PEPCK, hasbeen isolated (Perea and Gancedo, 1982). ThePCK1 gene coding PEPCK has been cloned byfunctional complementation of the mutant(Valdes-Hevia et al., 1989) and sequenced (Stucka

e-mail: kitamoto@abr.affrc.go.jp

CCC 0749–503X/98/100963–05 $17.50? 1998 by John Wiley & Sons, Ltd.

et al., 1988; Krautwurst et al., 1995). Theexpression of the PCK1 gene in S. cerevisiae isstrictly regulated and dependent on the carbonsource provided (Proft et al., 1995).

The yeast Kluyveromyces lactis, is closely relatedto S. cerevisiae with respect to DNA and predictedprotein sequences of the structural genes. How-ever, the physiologies of these two yeasts differconsiderably. A study of their galactose catabolism(Kuzhandaivelu et al., 1992) and fructose-1,6-bisphosphatase (Zaror et al., 1993) concludedthat S. cerevisiae demonstrates strong glucoserepression, whereas K. lactis displays only partialglucose repression in most strains. Understandingthe regulation of PEPCK expression in K. lactis isof considerable interest because it will help to

elucidate carbon metabolism in yeast.

964 . . .

In this paper, we present the cloning and se-quencing of the gene coding PEPCK from K.lactis. The deduced amino acid sequence has beencompared to those of other known PEPCKs.

MATERIALS AND METHODS

Strains, plasmids and mediumK. lactis IFO1267 (NRRL Y-1140; CBS 2359)

genomic DNA was used for cloning the geneencoding PEPCK (KlPCK1). S. cerevisiae CJM150(pck1 leu2 ura3) used for the complementationtest was kindly supplied by Dr Carlos Gancedo(Instituto de Enzimologia del C.S.I.C., Universi-dad Autonoma, Madrid). Bluescript II (KS+)(BSII; Stratagene Co. Ltd) was used as a cloningvector. YCp50 was used as a centromeric vector ofS. cerevisiae. The media were SD (0·67% yeastnitrogen base without amino acids, 2% glucose or0·5% calcium lactate and 36 ìg/ml leucine ifneeded) and YPD/YPG (1% Bacto yeast extract,2% Bacto peptone and 2% glucose/glycerol).

General DNA techniquesStandard DNA manipulations were performed

as described by Sambrook et al. (1989) using theEscherichia coli JM109 strain (Takara Shuzo).Genomic Southern hybridization (Southern, 1975)with the ECL direct nucleic acid labelling anddetection systems (ECL system; Amersham Inter-national plc) and colony hybridization (Walter andHoltke, 1992) with DIG-High Prime DNA label-ling and detection kit I (Boehringer Mannheim)was performed for cloning the KlPCK1 gene withPCK1, the PEPCK gene of S. cerevisiae, as theprobe. PCK1 was amplified by polymerase chainreaction (PCR; Sambrook et al., 1989) using gen-omic DNA of S. cerevisiae YNN27 as a templateand two primers selected to hybridize with the 5*and 3* ends of the Pck1 ORF (Krautwurst et al.,1995). Sequencing was performed on the deletionmutants prepared using a Kilo-Sequence DeletionKit (Takara Shuzo). The nucleotide sequences ofthe deletion mutants were analysed using the tech-nique of Sanger et al. (1977) and the Taq/DyePrimer and Terminator PRISM kits (Perkin ElmerCo. Ltd). Plasmid carrying the KlPCK1 was trans-formed into yeast by lithium acetate according toIto et al. (1983). The chromosomes of yeasts wereseparated using a contour-clamped homogeneouselectric field (CHEF) as described by Wesolowski-Louvel et al. (1996).

? 1998 John Wiley & Sons, Ltd.

Preparation of extracts and enzymatic assayThe S. cerevisiae strains were grown on SD

medium containing glucose for 2 days at 30)C on arotary shaker. The harvested cells were washed,transferred to the SD medium containing ethanolas the sole carbon source and shaken for 24 h. TheK. lactis strain was grown on YPD medium for24 h, the washed cells were transferred to the YPGor YPD medium and shaken for 13 h. Cell-freeextract preparation and PEPCK assay were carriedout as described by Perea and Gancedo (1982)with some modifications.

PEPCK activity was assayed at 30)C. The reac-tion mixture consisted of 25 ìmol imidazole,25 ìmol NaHCO3, 1 ìmol MnCl2, 1 ìmol glucose,1 ìmol reduced glutathione, 1 ìmol ADP,0·5 ìmol NADH, 0·1 ìmol phosphoenolpyruvate,80 n kat malate dehydrogenase and 40 n kathexokinase. The pH of the assay solution was 7·0.Protein was determined with a protein assay kit(BioRad Co. Ltd). To ascertain the ATP depen-dency of the PEPCK, crude protein of K. lactis wasprepared from cell-free extract by dialysis in 100volumes of cold 0·05 -potassium phosphatebuffer, 1 m-EDTA and 2 m-2-merceptoethanol,pH 7·0 for 18 h with three changes of buffer. Theenzyme activity was assayed as above with andwithout ADP.

RESULTS AND DISCUSSION

Cloning of K. lactis PEPCKA 1·6-kb fragment of the PEPCK gene of

S. cerevisiae (PCK1) was amplified by PCR. PCK1was used as a probe to isolate the K. lactis PEPCKgene. This fragment hybridized as a single band toK. lactis genomic DNA digested with EcoRI orHindIII in a Southern blot (Figure 1). The PCK1probe gave rather weak hybridization signalson K. lactis genomic digests in comparison withthat of S. cerevisiae. The hybridized EcoRI-digested DNA fragments were eluted from thegel and subcloned in BSII. A clone contained a4·8 kb insert (clone A3) was selected by colonyhybridization from the subclone.

Complementation of S. cerevisiae pck1To investigate the complementation of S. cerevi-

siae PCK1, plasmid YCp50 harboring the A3fragment was transformed into a PEPCK-deficientmutant of S. cerevisiae CJM150 in our laboratory.A mutant of S. cerevisiae lacking PCK1 is unable

Yeast, 14, 963–967 (1998)

965 .

to grow on medium containing lactate as a solecarbon source for gluconeogenic sources (Pereaand Gancedo, 1982). On the other hand, thetransformants could grow on SD medium contain-ing 0·5% calcium lactate (data not shown). PEPCKactivity of the cell-free extracts harvested in etha-nol media was measured. As shown in Table 1a,the PEPCK activity was recovered in the trans-formant. These results showed that the A3 frag-ment is functionally equivalent to PCK1 of S.cerevisiae, contains the PEPCK gene of K. lactiswith a promoter, and that the K. lactis promotercan function in S. cerevisiae. We named the geneKlPCK1.

? 1998 John Wiley & Sons, Ltd.

Mapping of the KlPCK1 geneThe chromosomes of K. lactis IFO1267 were

separated using CHEF. The A3 fragment hybrid-ized only with a band corresponding to chromo-some I in a Southern blot (data not shown). TheKlPCK1 gene has been localized on chromosome I,whereas the PCK1 gene of S. cerevisiae wasmapped on chromosome XI (Delgado andGancedo, 1992). K. lactis has only six chromo-somes. The sum of the molecular weights suggeststhat the genome size of K. lactis (12 Mbp) is aboutthe same as that of S. cerevisiae. K. lactis is closelyrelated to S. cerevisiae in ribosomal DNA se-quence revel. Wesolowski-Louvel and Fukuhara(1995) showed the latest genetic and physical mapof K. lactis. They revealed many rearrangements ofchromosomal organization in functionally equiv-alent genes between S. cerevisiae and K. lactis,whereas local order of genes can often be similar inboth strains. None of the K. lactis gene listed in themap has homology between any gene of chromo-some XI of S. cerevisiae. We need further infor-mation about the localization of the K. lactis geneon a map.

Figure 1. Genomic Southern hybridization of the PEPCKgene from strains K. lactis IFO 1267 and S. cerevisiae YNN27.The genomic DNAs were digested with HindIII, EcoRI orBamHI. Hybridization was carried out using the PEPCK gene(PCK1) of S. cerevisiae as a probe.

Table 1. Specific PEPCK activities of S. cerevisiae CJM150 (PEPCK-deficient) andtransformed CJM150 cell-free extract (a), crude protein from K. lactis IFO 1267 (b) andK. lactis IFO 1267 cell-free extract

StrainSpecific activity

(nmol/min per mg protein)

a S. cerevisiae CJM150 (PEPCK-deficient) 0·72S. cerevisiae CJM150 transformant 6·90

b K. lactis with ADP 51·3K. lactis without ADP 5·4

c K. lactis on glycerol 22·0K. lactis on glucose 1·8

Sequence analysis of KlPCK1The restriction pattern of the K. lactis A3 frag-

ment was different from that of S. cerevisiae (datanot shown). A Southern blot analysis showed thatan approximately 2·6 kb region in the middle ofthe A3 fragment had high homology to PCK1. Thenucleotide sequences of the part of A3 fragmentwhich showed high homology on hybridizationwere analysed. The ORF sequence was 1629 bp,the deduced amino acid sequence was 543 aminoacids (Figure 2), and the molecular weight of theproduct calculated from the predicted amino acidsequence was 60,325 Da.

Yeast, 14, 963–967 (1998)

966 . . .

Figure 2. The nucleotide sequence and deduced amino acid sequence of KlPCK1. The residue of the first ATG codonin the ORF is numbered 1. The consensus sequences conserved in the ATP-dependent PEPCK (Linss et al., 1993) areunderlined and labeled a–d. (a) Unknown structural sequence; present in all, (b) phosphate-binding region inATP-using proteins, (c) protein binding site for divalent or transition metal ion and (d) binding site for adenine inATP-using proteins. The nucleotide sequence of KlPCK1 has been submitted to the DDBJ/GenBank/EMBL databank with accession number U88575.

Comparison of deduced PEPCK amino acidsequences of K. lactis, S. cerevisiae and otherorganisms

Sequences were analysed using the MP searchprogram (Intelli Genetics Inc.). The DNA se-quence and deduced amino acid sequence showed76% and 84% homology to those of S. cerevisiae,respectively. The PEPCKs found in plants andmicroorganisms are usually ATP-dependent,whereas those found in animals are almost exclu-sively GTP-dependent (Alvear et al., 1992). Thededuced amino acid sequence of ATP-dependentPEPCKs reported in GenBank was very similar tothat of KlPCK1. The ATP-dependent PEPCKs,phosphate and adenine-binding regions that arecharacteristic of ATP-using proteins are clearlyidentifiable (Linss et al., 1993). The four consensussequences in KlPck1 (underlined segments inFigure 2) were also highly conserved. These datashow that KlPck1 is very similar to Pck1. Further-more, KlPck1 showed no significant homologieswith GTP-dependent PEPCKs as described byStucka et al. (1988). The PEPCK activity of thecrude enzyme from K. lactis IFO 1267, harvested

? 1998 John Wiley & Sons, Ltd.

in glycerol, was very low in the absence of ADP(Table 1b), indicating that it is the ATP-dependenttype. These data coincide with the amino acidsequence homology data.

Occasionally the isolation of K. lactis homo-logues with transcriptional regulators by comp-lementation of S. cerevisiae mutants can be clonedtogether (Wesolowski-Louvel and Fukuhara,1995). We showed the functional complementationof pck1 of S. cerevisiae by KlPCK1 including thepromoter region. However, the promoter region ofS. cerevisiae PCK1 gene (Mercado and Gancedo,1992) and the nucleotide sequence 390 bp up-stream of the first ATG of the KlPCK1 codingsequence shared no homology. The PEPCK ac-tivity of K. lactis on different carbon sources wasmeasured (Table 1c). The inactivation rate byglucose was 92%. This indicates that the PEPCKof K. lactis IFO1267 is repressed by glucose. Thisresult supports the conclusion that the regulationmechanism of KlPCK1 expression is different fromthat of PCK1. Further work is needed to assess therole of the promoter in the transcription ofKlPCK1.

Yeast, 14, 963–967 (1998)

967 .

Conclusions(1) The gene encoding K. lactis PEPCK was

cloned and sequenced. The nucleotide sequenceand deduced amino acid sequence showed 76%and 84% homology to S. cerevisiae PCK1 respect-ively. (2) KlPCK1 and its promoter region comp-lement the S. cerevisiae pck1 mutation. (3) PEPCKof K. lactis IFO1267 is repressed by glucose.

ACKNOWLEDGEMENTS

This research was supported by a grant in 1994from the National Grassland Research Institute,Japan, and by a grant in 1995 from the Scienceand Technology Agency, Japan. We thank DrHiroshi Fukuhara (Institut Curie, Centre Univer-sity, France) and Dr Koji Nakamura (TsukubaUniversity, Japan) for helpful discussions.

REFERENCES

Alvear, M., Encinas, M. V., Kemp, R. G., Latshaw,S. P. and Cardemi, E. (1992). ATP-dependentSaccharomyces cerevisiae phosphoenolpyruvate car-boxykinase: isolation and sequences of a peptidecontaining a highly reactive cysteine. Biochim.Biophys. Acta 1119, 35–38.

Delgado, M. A. and Gancedo, C. (1992). Mapping ofthe PCK1 gene encoding phosphoenolpyruvate car-boxykinase on chromosome XI of Saccharomycescerevisiae. FEMS Microbiol. Lett. 92, 125–128.

Ito, H., Fukada, Y., Murata, K. and Kimura, A. (1983).Transformation of intact yeast cells treated with alkalications. J. Bacteriol. 153, 163–168.

Krautwurst, H., Encinas, M. V., Marcus, F., et al.(1995). Saccharomyces cerevisiae phosphenolpyruvatecarboxykinase: Revised amino acid sequence, site-directed mutagenesis, and microenvironment charac-teristics of cysteines 365 and 458. Biochemistry 34,6382–6388.

Kuzhandaivelu, N., Keith Jones, W., Martin, A. K. andDicson, R. C. (1992). Mol. Cell. Biol. 12, 1924–1931.

Linss, J., Goldenberg, S., Urbina, J. A. and Amzel,L. M. (1993). Cloning and characterization of thegene encoding ATP-dependent phosph-enol-pyruvate

? 1998 John Wiley & Sons, Ltd.

carboxykinase in Trypanosoma cruzi: comparison ofprimary and predicted secondary structure with hostGTP-dependent enzyme. Gene 136, 69–77.

Mercado, J. J. and Gancedo, J. M. (1992). Regulatoryregions in the yeast FBP1 and PCK1 genes. FEBSLett. 311, 110–114.

Perea, J. and Gancedo, C. (1982). Isolation and charac-terization of a mutant of Saccharomyces cerevisiaedefective in phosphoenolpyruvate carboxykinase.Arch. Microbiol. 132, 141–143.

Proft, M., Grzesitza, D. and Entian, K.-D. (1995).Identification and characterization of regulatoryelements in the phosphenolpyruvate carboxykinasegene PCK1 of Saccharomyces cerevisiae. Mol. Gen.Genet. 246, 367–373.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNAsequencing with chain-terminating inhibitors. Proc.Natl. Acad. Sci. USA 74, 5463–5467.

Southern, E. M. (1975). Detection of specific sequencesamong DNA fragments separated by gel electro-phoresis. J. Mol. Biol. 98, 503–517.

Stucka, R., Valdes-Hevia, M. D., Gancedo, C.,Schwarzlose, C. and Feldmann, H. (1988). Nucleotidesequence of the phosphoenolpyruvate carboxykinasegene from Saccharomyces cerevisiae. Nucl. Acids Res.16, 10,926.

Valdes-Hevia, M. D., Guerra, R. and Gancedo, C.(1989). Isolation and characterization of the geneencoding phosphenolpyruvate carboxykinase fromSaccharomyces cerevisiae. FEBS Lett. 258, 313–316.

Walter, T. and Holtke, H.-J. (1992). Colony and plaquehybridization with the digoxigenin system. Colloquium2, 8–9.

Wesolowski-Louvel, M. and Fukuhara, H. (1995). Amap of the Kluyveromyces lactis genome. Yeast 11,211–218.

Wesolowski-Louvel, M., Breunig, K. D. and Fukuhara,H. (1996). In Wolf, K. (Ed.), Nonconventional Yeastsin Biotechnology. Springer-Verlag, Berlin, pp. 140–201.

Zaror, I., Marcus, F., Moyer, D. L., Tung, J. andShuster, J. R. (1993). Fructose-1,6-bisphosphatase ofthe yeast Kluyveromyces lactis. Eur. J. Biochem. 212,193–199.

Yeast, 14, 963–967 (1998)