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PYRUVATE

CARBOXYLASE: ENCODING

Michelle

AND

E. Walker*,

IDENTIFICATION

OF TWO

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GENES

ISOENZYMES

Dale L. Val*, Manfred Rohde#, and John C. Wallace*

Rodney

J. Devenish+,

*Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5001, Australia #Bereich Mikrobiologie, Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig, Germany +Department of Biochemistry, Received

February

13,

Monash University, Cklyton, Victoria 3168, Australia

1991

m: In Saccharomyces cerevisiae, pyruvate carboxylase [EC 6.4.1.11 has an important anapleroticrole in the production of oxaloacetatefrom pyruvate. We report herethe existenceof two pyruvate carboxylase isozymes, which are encodedby separategeneswithin the yeast genome. Null mutants were constructed by one step gene disruption of the characterisedPYC genein the yeast genome.The mutantswere found to have lo-20% residual pyruvate carboxylase activity, which was attributable to a protein of identical size and immunogenically related to pyruvate carboxylase. Immunocytochemical labelling studieson ultrathin sectionsof embeddedwhole cells from the null mutants showedthe isozyme to be located exclusively in the cytoplasm. We have mappedthe genesencoding both enzymes and shown the previously characterisedgene,designatedPYCI, to be on chromosomeVII whilst PYC2 is on chromosomeII. 0 1991Academic Press,Inc.

Pyruvate carboxylase [EC 6.4.1.11, a biotin-dependent enzyme present in a wide variety of organisms,hasan important anapleroticfunction in replenishingthe intermediatesof the tricarboxylic acid (TCA) cycle by catalysing the carboxylation of pyruvate to form oxaloacetate (for review see [l]). In vertebrates, pyruvate carboxylase is a mitochondrial enzyme located in the mitochondrial matrix ] l] whilst in filamentousfungi [2,3] and yeast [4,5] it has a cytosolic location. In both groups of organisms,the enzyme is composedof four subunitsin a tetrahedral arrangement[6,7], with eachsubunit having a prosthetic biotin group covalently attached[81. To facilitate studieson the structure and function of pyruvate carboxylase, we have isolated the gene from yeast [9,10]. On the basisof sequencecomparisonswith other biotin enzymes, we have tentatively assignedfunctions to regionsof the protein involved in the two partial reactions [lo]. In this study, we have constructedtwo null mutantsin which either most of the previously characterisedPYC gene,designatedPYCI, wasreplacedwith the functional yeast LEU2 gene, or the yeast HIS3 genewas insertedinto the DNA sequenceencodingthe Abbreviations: DTT - DL-Dithiothreitol; EDTA - Ethylenediaminetetraacetate (sodium); PMSF - Phenylmethylsulphonylfluoride; PYC - Pyruvate carboxylase; SDS - Sodium dodecyl sulphate. 0006-291X/91 Copyright All rights

$1.50

0 1991 by Academic Press. Im.. of reproduction in any ,form reserved.

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ATP/HC03binding domain [lo]. We demonstrate that the disruption of this PYCI gene reveals the presence in yeast of a second gene (PYC2 ), the protein product of which we have identified by biochemical analysis and immuno-electron microscopy . Materials

and Methods

Strains, media and transformation procedures: Saccharomyces cerevisiae strains DBY746 (MATa, his3,leu2, ura3, trpl), YPH80 (MATa, ura3,lys2, ade, his7, trpl) and YPH149 (MATa, ura3, lys.2, ade, his’/, trpl) were used. Strain YPH149 carries a proximal RAD2 chromosome VII fragment (URA3 +) and a distal RAD2 chromosome VII fragment (TRPl’). All strains were grown in glucose minimal and YPD media as described by Walker ef al. [ 111. Putative pyc mutants were selected on their inability to grow on glucose minimal medium unless supplemented with 2.6 mM L-aspartate. Escherichia cofi strains ED8799, DHSa, and dam strain GM1 19 were used for plasmid propagation. E. coli transformation was according to Mandel and Higa [ 121. Nucleic acid manipulations: Restriction enzymes, T4 DNA ligase and Klenow fragment were purchased from Bresatec Ltd., Adelaide. DNA manipulations were performed according to Maniatis et al. [13]. Radioactive DNA probes were prepared using oligolabelling kits obtained from Bresatec Ltd., Adelaide. Plasmid DNA preparations were made by the alkaline lysis method [14]. Genomic DNA was isolated from yeast grown overnight in lOm1 YPD using a small scale version of the method according to Cryer et al. [ 151. PYC gene disruption: The pyc yeast strain MW21.3 (MATU, his3, trpl, ura3, pycl::LEU2) was constructed by the one-step gene disruption method [16]. The 4kb HindI11 PYCI fragment [lo] was inserted into pSP64 [ 171, yielding the plasmid pMWl.The 2.4kb BglII PYCI fragment (containing the promoter and ATP domain coding sequences) was excised from pMW1, and replaced with the 3kb BgiII L.EU2 fragment from YEpl3 [ 181 to give the recombinant plasmid pMW3. The plasmid was cut with HindIII, and the digest used directly to transform the haploid S. cerevisiae strain DBY746 to LEU+. A second null mutant, MW15.2.2 (MATa, pycl ::HIS3,trpl, ura3, feu2) was constructed by inserting the intact HIS3 gene at codon Va1175 of the ATP/HCOj- binding domain. The 1.76 kb BamHf fragment from YEp6 1191 containing the intact HIS3 gene was inserted at the BclI site of pMW1, yielding the recombinant plasmid pMW13. The plasmid was cut with BamHI and the digest used to transform the strain DBY746 to HIS+. The disruption of the chromosomal PYCI gene in the strains MW21.3 and MW15.2.2 was confirmed by their inability to utilize glucose unless supplemented with 2.6mM aspartate (demonstrated in a plate assay for growth over 2 days at 30°C), and by Southern blot hybridisation. Chromosomal mapping of pyruvate carboxylase genes: Yeast chromosomes from strains DBY746, MW21.3, YPH80 and YPH149, were prepared in agarose plugs as described by Schwartz and Cantor [20]. The chromosomes were separated by pulse field gel electrophoresis in a 1% agarose/0.5x TBE (45.5mM Tris-borate, 45.5mM boric acid, 1mM EDTA) gel using the Biorad Chef-DRII system. After electrophoresis, the DNA was transferred onto Nytran (Schleicher and Schuell), and hybridised with either the 2.4kb BglII PYCI and 1.04 kb BamHI-HincII PYCI fragments as probes for pyruvate carboxylase[lO], or with chromosome specific probes for chromosomes XV (HIS3; [19]), XIV (centromere sequences; [21), VII (CUPZ; [22]) and II (LYS2; [231). Immunocytochemical localisation of pyruvate carboxylase: Whole yeast cells were fixed with a solution containing 0.2% (v/v) glutnraldehyde and 1% (v/v) formaldehyde in PBS (0.1 M phosphate-buffer, 0.9% (w/v) NaCl, pH 6.9) for 1 h on ice. Embedding in the low temperature resin Lowicryl K4M was carried out according to Carlemalm et al. [24] and Roth et al. [25] with some modifications. In brief: dehydration was achieved with a graded series of ethanol; the infiltration steps with the resin-ethanol mixtures at each step were carried out at -35’C for 1 day; the infiltration with the pure resin was done over 3 days with a complete change of the resin every day at -35’C. Polymerization was performed using UV-light (366nm) for 1 day at -35’C and for 2 days at room temperature. Colloidal gold-particles were 1211

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prepared for immunocytochemical labelling according to Lim et al. [4]. Electron micrographs were taken with a Zeiss EM 1OB or CEM 902 electron microscope at calibrated magnifications and at an acceleration voltage of 80kV. methods: Cell-free extracts were prepared from 20-24 hr yeast cultures grown in 1OOmlof YPD. The cells were pelleted, washed in lysis buffer (100mM Tris HCl pH 7.2, 1OmMMgC12, 1mM EDTA, 1mM PMSF, 1mM DTT), and vortexed for 2 minutes with glass beadsin fresh lysis buffer. Cell debris wasremoved by centrifugation at 13,000x g for 10 min. Pyruvate carboxylase assayswere carried out on the equivalent of 2ml of cell culture as described by Lim et al. [4]. Protein content was determined by the Bradford method [26]. Western analysiswasperformed accordingto Lim et al. [4]. Protein

Results

and

Discussion

In this study we have constructed the yeast null mutants, MW21.3 and MW15.2.2 by disrupting the chromosomalPYCl geneof DBY746 asdescribedin the Materials and Methods section. Figure 1 showsa Southern blot of the digested DNA probed with the 2.4kb Bgl II PYCI fragment (Fig. 1A - Hind111and BglII digests) and the 1043bp HincII-BamHI PYCl fragment encoding the “pyruvate domain” [lo] (Fig. 1B - BglII digest). The disruption of the cloned PYCl gene can be seenby the disappearanceof the 2.4kb BglII and 4kb Hind111 fragments in MW21.3 when probed with the 2.4kb BglII fragment (which has beenreplaced with the LEU2 gene fragment - seeMaterials and Methods). In the caseof MW15.2.2, the insertion of the HIS3 gene results in two HindIII fragments (2.2kb and 3.3kb) and two BglTI fragments (1.9kb and 1.4kb) due to the presenceof Hind111and BglII sitesin the HIS3 gene [16]. As the 3’ region of the cloned PYCI gene was not mutated, we observe the expected 2.lkb BgAI fragment in all three strainswhen probedwith the “pyruvate domain” fragment

kb 21

3 -

X9= 43

-

23 20

-

056

Fig. 1. PYCZ

-

Southern blot analysis of genomic gene on the yeast chromosome.

DNA:

One-step

disruption

of the

GenomicDNA from theparentalPYC+strainDBY746, the LEU2 transformant MW21.3 and the HIS3 transformant MW15.2.2 was cut with either Hi&III or BgnI and the digests were separated on a 0.8% agarose gel in TBE (Tris-borate) buffer. The DNA was denatured, transferred to Nytran and fixed by UV cross linking using the Stratalinker UV crosslinker 1800 apparatus (Stratagene). The filters were prehybridised overnight at 42°C and subsequently hybridised with A the 2.4kb EglII PYCI fragment and B the 1.043kb HincIIBarnHI “pyruvate domain” fragment (48 hours at 42’C) [ 131. The filters were washed at 50°C in 0. lx SSC [lSmM NaCl, 1.5mM sodium citrate pH7]/0.1% SDS, before autoradiography. The migration of the Hind111 cut k DNA standards (23.1 kb, 9.4 kb, 6.5 kb, 4.3 kb, 2.3 kb, 2.0 kb, 0.56 kb) are shown. The symbol (F) indicates a fragment containing the PYCZ gene. 1212

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Table

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1. Pyruvate

AND

carboxylase

BIOPHYSICAL

activity

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in yeast cell extracts

Enzyme activity

Strains

(mU/mg f SEM)

Avidin-sensitive

DBY746 MW21.3 MW15.2.2

45.89 + 1.62 4.54 + 0.49 8.34 f 1.01

Pyrnvate-dependent

DBY746 MW21.3 MW15.2.2

43.40 It 2.20 4.14 rt 0.74 9.01 + 1.2

Antibody-specific

DBY746 MW21.3 MW15.2.2

43.15 + 2.58 4.55 + 0.39 9.45 f 0.75

Enzyme units (mU/mg of protein at 3O’C) were determined in duplicate in four different crude ceil extracts made fromeither wild type ormutant yeast grown in YPD. Values given are the mean of measurements + the standard error of the mean (SEM). Pyrnvate carboxylase activity was measured using the 14CO~ incorporation assay [4] either in the presence or absence of 48Qglml avidin (avidin-sensitive units), 1OmM pyruvate (pyrnvate-dependent units), 5ng polyclonal antibody (antibody-specific units). An unexpected observation was the hybridisation of the probes to distinct bands (marked with an [, I), which do not correspond to the known restriction map of the complete PYCI gene [lo]. These bands do not arise from incomplete digestion of the DNA (as the results are repeatable and unaffected by incubation time or enzyme concentration), but rather, they suggest the presence of a second gene encoding The gene disruption method when targeted

a similar isozyme, or a pseudogene. to a specific gene locus in a haploid

cell,

gives rise to a null mutant lacking the endogenous protein normally expressed in the wild-type strain [ 111. However, we have detected a significant amount of pyruvate carboxylase activity (lo-20%) in the null mutants, as shown in Table 1. Western blot analysis (Fig. 2) showed the kDa

f-l

YPC

DBY

205-

21

15

YPC

DBY

21

(-iJ**

15

YPC

DBY

_I.

21

15

._-..,.

11697.466-

45-

29-

Caomassie

anti-YPC

F&&

avidin-AP

Western analysis of cell extracts of wild-type and pyc yeast. Equal amounts of total protein extracts from the parental strain DBY746 (DBY), the null mutants MW21.3 (21), MW15.2.2 (15) and purified yeast pyruvate carboxylase (YPC) were electrophoresed and (a) stained with Coomassie blue, (b) transferred to nitrocellulose and probed with anti-YPC antibody followed by goat anti-rabbit IgG conjugated to alkaline phosphatase, and (c) transferred to ninocellulose and probed with avidin conjugated to alkaline phosphatase. Molecular weight markers (M) were carbonic anhydrase (29 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (97.4 kDa), P-galactosidase (116 kDa) and myosin (205 kDa). Biotin enzymes are acetyl CoA carboxylase (205 kDa), pyruvate (47 kDa). carboxylase (130 kDa) and urea carbxylase 1213

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:

Fig. 3. Immunocytochemical the protein A-gold labelling

localisation protocol.

of yeast pyruvate

carboxylase

using

Ultrathinsections of low temperature embedded wholeyeastcellsfrom MW21.3were treatedwith anti-yeastPC antibody(A,B,C; A andB 150pg IgG/ml, C, 75pg IgG/ml) followedby proteinA-gold complexes(15 nmin size,OD520nm = 0.02)andpoststained with uranylacetate.PanelD depictsa controlexperiment.Thesectionwasincubatedwith non-specificIgG antibodies (normalrabbitserum)followedby theproteinA-gold complexes. Thedarkdotsareindicativefor pyruvatecarboxylasewhichis exclusivelylocatedin the cytoplasm.Thecontrolexhibitsfew gold-particles thusdemonstrating the specificityof the labellingobtainedin paneisA-C. Barsrepresent0.5km in A andD, 0.2 pmin B andC. CW, cellwall; G, gold-particle;M, mitochonclrion;N, nucleus;V, vacuole. mutantsto exhibit significant levels of the pyruvate carboxylase protein, which is identical in size to purified pyruvate carboxylase, is biotinylated and immunologically related to the purified enzyme. Pyruvate carboxylase in the mutants was quantitated by densitometric scanning of the Western blots (Molecular Dynamics Computer Densitometer 300A), and shown to be present at only 38% (MW15.2.2) and 41% (MW21.3) of the wildtype level. RNasemapping of yeast total RNA showed specifically that there was no transcription from the disrupted PYCI genein the mutantsMW21.3 and MW15.2.2 (data not shown). Figure 3 shows electron micrographs of ultrathin sections of low temperature embeddedwhole cells from the null mutant MW21.3 (which lacks the PYCl product) after incubation with rabbit anti-yeastpyruvate carboxylasepolyclonal antibodiesandprotein A-gold complexes. The presenceof dark grains only in the cytosol demonstratesthe existence of a pyc

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BIOCHEMICAL

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d

C

I

2

3

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4

I

0

PYCl “N-terminal”

CUP2 - Chr VII

e

2

3

4

-LO

HIS3 - Chr XV

h

PYC I

“Pyruvate”

LYS2 - Chr II

I

CEN - Chr XIV

Fig. 4. Chromosomal localization of pyruvate carboxylase genes (PYCI and PYC2) in wild-type and null mutant yeast strains. (a) Intact chromosomal DNA was prepared from 1. DBY746, 2. MW21.3, 3. YPH80, 4. YPH149 and was separated by Chef pulse field electrophoresis as previously described. The order of the chromosomes are indicated with the exact positions referring to the separated chromosomes from strain YPH80 in Lane 3. The DNA was transferred to Nytran and subsequently probed in separate experiments with the following probes, (b) 2.4kb Bg[II N-terminal PYCl fragment, (c) 2.lkb DraI CUP.2 fragment (chromosome VII), (d) 1.76kb BamHI HIS3 fragment (chromosome XV), (f) 1.04kb BamHI-Him11 PYCl “pyruvate domain” fragment, (g) 6.9kb EcoRI-PstI LYS2 fragment (chromosome II), (h) 1.4kb EcoRI-Hi&III CEN fragment (chromosome XIV). Gel (a) was reproduced in the figure as gel (e) to allow for alignment of autoradiographs of the gel after hybridisation.

isoform of pyruvate carboxylase synthesizedfrom a gene distinct from the previously cloned PYCl gene [lo]. As the mitochrondrial inner membraneis impermeableto oxaloacetate the oxaloacetate produced by this enzyme in yeast must be transported to the mitochondrialmatrix by a shuttlemechanisminvolving the conversionof oxaloacetateto malate by malatedehydrogenase[EC 1.1.I .37] [27]. We have used chromosomeblot analysisto map the PYC genesto chromosomeVII (PYCl) and II (F’YC2 >. Yeast chromosomeswere isolated from four strains; the null mutant MW21.3, its parent strain DBY746, and two other independent PYC+ strains,YPH80, and YPH149 which is a strain in which chromosomeVII is split in two at the RAD2 locus [28].

second cytosolic

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The separated chromosomes bound on Nytran membrane were probed with the 2.4kb BgllI PYCl “N-terminal” fragment (Fig. 4b). Two chromosomal bands from strains DBY746, PYCl probe [lo]. The strongest YPH80 and YPH149 hybridised to this “N-terminal” hybridisation signal, which corresponds to the PYCI gene, aligns with the position of the chromosome XV/VII doublet in DBY746 and YPHSO, while its position in the lane containing YPH149 chromosomes clearly demonstrates that the PYCI gene is on chromosome VII proximal to the RAD2 gene. Further confirmation of this assignment was obtained by reprobing the same membrane with the CUP2 gene as a marker for chromosome VII (Fig. 4~) and the HIS3 gene as a marker for chromosome XV (Fig. 4d). Alignment of the figures 4a-4d revealed that the PYCI gene, like the CUP2 gene, is located on the RAD2.P fragment of chromosome VII, rather than on chromosome XV (the band hybridising to the HIS3 gene) which in strains other than YPH149 co-migrated with chromosome VII. The chromosome XV/VII doublet from MW21.3 did not hybridise to the “N-terminal” PYCZ probe as the DNA sequences complimentary to this probe were removed in the construction of this null mutant. The weaker hybridisation at chromosome II (Fig. 4b) represents a second gene (PYC2 ) which has some sequence similarities to PYCl. To ensure that the hybridisation signals produced by the “N-terminal” probe were indeed due to pyruvate carboxylase genes, the membrane was re-probed with the “pyruvate domain” PYCI probe (Fig. 4f) cormdining sequences which appear to be entirely unique to pyruvate carboxylase [lo]. As expected, the two hybridisation signals align with those observed for PYCI and PYC2 in figure 4b. In addition, a band is observed for PYCI (chromosome VII) with the null mutant MW21.3 as the “pyruvate domain” is intact in this mutant. The physical mapping of PYC2 to chromosome II was confirmed by re-probing the filter with the LYS2 gene as a probe for chromosome II (Fig. 4g), and centromere sequences from chromosome XIV (Fig. 4h). Alignment of the figures 4e-4h revealed that the PYC2 gene was located on chromosome II and not on the closely migrating chromosome XIV. Isolation of the PYC2 gene, now in progress, will enable us to produce double null mutants to use as hosts for genetically engineered forms of PYC gene(s) in structure and function studies.

Acknowledgme

ts: This work was supported by an Australian Research Council grant (A0 8932217) to”3.C.W. We thank Dr. Phillip Hieter (John Hopkins University Baltimore USA) for his gift of the Saccharomyces cerevisiue strains YPH80 and YPH149. D.L.V. is the recipient of an Australian Postgraduate Research Award.

Peferenw 1. 2. 3. 4. 5. 6. 7.

Wallace, J.C. (1985) in Pyruvate Carboxyluse (Keech, D.B., and Wallace, J.C. eds) ~5-63, C.R.C. Series in Enzyme biology, C.R.C. Press, Boca Raton, U.S.A. Osmani, S.A. and Scrutton, M.C. (1983) Eur. J. Biochem. 133, 551-560 Osmani, S.A. and Scrutton, M.C. (1985) Eur. J. Biochem. 147, 119-128 Lim, F., Rohde, M., Morris,. C.P. and Wallace, J.C. (1987) Arch. Biochem. Biophys. 258, 259-264 van Urk, H., Schipper, D., Breedveld, G.J., Mak, P.R., Scheffers, W.A. and van Dijken, J.P. (1989) Biochem. Biophys. Acta. 992, 78-86 Mayer, F., Wallace, J.C. and Keech, D.B. (1980) Eur. J. Biochem. 112, 265-272 Rohde, M., Lim, F. and Wallace, J.C. (1986) Eur. J. Biochem. 156, 15-22 1216

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Barden, R.E., Fung, C-H., Utter, M.F. and Scrutton, M.C. (1972) J. Biol. Chem. 247, 1323-1333 Morris, C.P., Lim, F. and Wallace, J.C. (1987) Biochem. Biophys. Res. Commun. 145, 390-396 Lim, F., Morris, C.P., Occhiodoro, F. and Wallace, J.C. (1988) J. Biol. Chem. 263, 11493-11497 Walker, M.E., Valentin, E. and Reid, G.A. (1990) Biochem. J. 266, 227-234. Mandel, M. and Higa, A. (1970) J. Mol. Biol. 53, 159-162 Mania& T., Fritsch, E.F. and Sambrook, J. (1982) in Molecular Cloning - A Laboratory Manual, 8th printing, Cold Spring Harbor Birnboim, H.C. and Doly, J. (1979) Nut. Acids Res. 7, 1513-23 Cryer, D.R., Eccleshall, R. and Marmur, J. (1975) in Methods in Cell Biology, (D.M. Prescott, Ed.), Vol. 12, ~39, Academic Press, New York Rothstein, R.J. (1983) Methods Enzymol. 101, 202-11 Krieg, P.A. and Melton, D.A. (1984) Nut. Acids Res. 12, 7057-7070 Broach, J.R., Strathen, J.N. and Hicks, J.B. (1979) Gene 8, 121-133 Struhl, K., Stinchcomb, D.T., Schere, S. and Davis, R.W. (1979) Proc. Nafl.Acad. Sci. U.S.A. 76, 1035-1039 Schwartz, D.C. and Cantor, C.R. (1984) Cell 37, 67-75 Carle, G.F., and Olsen, M.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3756-3760. Welch. J., Fogel, S., Buchman, C. and Karin, M. (1989) EMBO J. 8, 255-260 Eibel, H. and Philippsen, P. (1983) Mol. Gen. Genet. 191, 66-73 Carlemalm, E., Garavito, M. and Villiger, W. (1982) J. Microscopy 126, 123-143. Roth, J., Bendayan, M., Carlemalm, E., Villiger, W. and Garavito, M. (1981) J. Histochem. Cytochem. 29, 663-669. Bradford, M.M. (1976) Anal. Biochem. 72, 248-54 Dijkema, C. and Visser, J. (1987) Biochem. Biophys. Acta, 931, 311-319. Vollrath, D., Davis, R.W., Connelly, C., and P. Hieter (1988) Proc. Natf. Acad. Sci. USA 85, 6027-603 1.

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Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes.

In Saccharomyces cerevisiae, pyruvate carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from pyruvate. We r...
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