Eur. J. Biochem. 191,769 - 774 (1 990) FEBS 1990

Molecular cloning of the gene for the E l a subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae H. Yde STEENSMA',3, Lennart HOLTERMAN '.*,Iris DEKKER', Cees A. van SLUIS' and Thibaut J. WENZEL3 Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands Laboratory of Molecular Genetics and Department of Cellbiology and Genetics, Leiden University, The Netherlands (Received October 19, 1989/February 23, 1990) - EJB 89 1262

The Eln and E1B subunits of the pyruvate dehydrogenase complex from the yeast Succharomyces cerevisiue were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector Agtl 1. The gene encoding the Elcl subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identity of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-Ela serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-Ela serum in haploid cells. In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human Elm peptide. We propose to call the gene encoding the Elcr subunit of the yeast pyruvate dehydrogenase complex PDAI. Screening of the Agtll library using the anti-Elp serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.

The yeast Succharomyces cerevisiae is widely used for the production of a large variety of compounds. These range from simple metabolites like ethanol to heterologous gene products or even the complete yeast cells. A disadvantage of this yeast species is the often unwanted production of ethanol, even at relatively low glucose concentrations in the presence of sufficient oxygen, the so-called Crabtree effect [l]. Fed-batch cultivation under sugar limitation and fermentor design may efficiently suppress the Crabtree effect in large-scale fermentations. Alternatively, genetic engineering may be used to reroute metabolic fluxes and reduce unwanted byproducts. It is thought that ethanol production depends o n the internal pyruvate concentration [ 2 -61. To investigate the possibilities of suppressing the Crabtree effect by metabolic pathway engineering, we have been studying the genetics of pyruvate metabolism in yeast. Pyruvate is an intermediate in both anabolic and catabolic pathways. In anabolism, alanine synthesis, replenishment of the citric acid cycle with oxaloacetic acid and formation of CoASAc, the building block for lipid synthesis, proceed via pyruvate. The enzymes involved are alanine 2-oxoglutarate transaminase, pyruvate carboxylase and the enzymes of the Correspondence to H . Y . Steensma, Clusius Laboratory, Leiden University, Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands. Abbreviations. O FAGE, orthogonal field-alternation gel electrophoresis. Enzymes. Alanine 2-oxoglutarate transaminase (EC 2.6.1.2); pyruvate carboxylase (EC 6.4.1.I); pyruvate decarboxylase (EC 4.1.1.1); alcohol dehydrogenase (EC 1.1.l.l); pyruvate dehydrogenase (EC 1.2.4.1); lipoate acetyltransferase, E2 (EC 2.3.1.12); dihydrolipoamide dehydrogenase, E3 (EC 1.8.1.4); 2-oxoglutarate dehydrogenase (EC 1.2.4.2); branched-chain 2-0x0-acid dehydrogenase (EC 1.2.4.4).

pyruvate dehydrogenase complex. Although mutants in the gene for pyruvate carboxylase (PYC) have been reported, these mutants were later shown to have the same enzyme levels as the parental strain [7] and the phenotype of pyc- mutants is still unclear. Recently, the gene has been cloned and sequenced [8, 91. In catabolism, pyruvate is an intermediate in ethanol production and in energy generation via the citric acid cycle. The conversion into ethanol via acetaldehyde has been studied extensively both at physiological and genetic levels. The genes of the enzymes involved, i.e. pyruvate decarboxylase and alcohol dehydrogenase, have been cloned and characterized [lo- 161. Pyruvate enters the citric acid cycle via CoASAc. The reaction is performed by a multienzyme complex, the pyruvate dehydrogenase complex. This complex is highly conserved among eukaryotes and consists of at least three catalytic subunits. E l is the pyruvate dehydrogenase enzyme, which has two distinct subunits, Elcl and Elp. E2 is a lipoate acetyltransferase and E3 is dihydrolipoamide dehydrogenase. A minor component, called protein X, has been identified in the bovine, rat and human complexes. A protein kinase which copurifies with the complex and a phosphatase regulate the activity of the mammalian complex by (de)phosphorylation of the Elcr subunit [17, 181. The pyruvate dehydrogenase complex is one of three multienzyme complexes which have an analogous composition. Both 2-oxoglutarate dehydrogenase and branchedchain 2-0x0-acid dehydrogenase have four components. The E3 subunit is present in all three complexes and is encoded by a single gene in the yeast genome. The E l and E2 subunits are specific for each complex. More details on the biochemistry

770 Table 1. Yeast strain.,

Strain

Gcnotypc

Source 01 reference

AB972 W303-1B X3402-15C YS59 YS60 YS62

M A T E rho’ t r p l M A T x ade2-1 his3-11, 15 leu2-3, I12 trpl-1 ura3-I MATa A D E l I A D E l a d d arg1 arg4 gull his5 leu1 met2pet17 uru3 MATcl FLOI his4-519 leu2-3,112 uru3-52 MATulMATx FLOl/Jlol his4-519/his4-519 leu2-3,1121+ trpl-789/+ ura3-52/ura3-52 MATu FLOI hid-519 leu2-3,112 trpl-789 ura3-52

~311 R Rothstein [321 thls study this study this study

and the regulation of the pyruvate dehydrogenase complex may be found in recent reviews [19, 201. The genes coding for subunits of several mammalian complexes, including the human complex have already been cloned. Elcr [21 -231, E l P [22, 241, E2 [25], E3 [26]. In yeast, the L P D l gene, which encodes the E3 subunit, and the gene for the E2 subunit have been isolated and sequenced [27 - 301. To be able to study the yeast pyruvate dehydrogenase complex in more detail, we decided to isolate the genes of the remaining subunits. As a first approach we attempted to isolate Pdh- mutants. These attempts were unsuccesful, even in a pyruvate-decarboxylase-negative (Pdc-) mutant. The rationale of the latter approach was to prevent the formation of CoASAc via acetaldehyde and acetate, which would jeopardize the selection procedure, i.e. selection of mutants that were not able to grow on glucose but still grew on ethanol. In this contribution, we report the purification of the Elm and E1P subunits of the yeast pyruvate dehydrogenase complex and the isolation of the gene encoding the Elcl subunit from an expression library containing S . cerevisiae genomic DNA. MATERIALS AND METHODS Strains

Cell-free extract

Extracts for immunoblotting were obtained from cells grown in YEPGal(1 YOBacto-yeast extract, 2% Bactopeptone, 1 % D-galactose). Cells from 100 ml culture were harvested, resuspended in 1 ml cold buffer (50 mM K 2 H P 0 4 pH 7.8, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% Triton X-100) and vortexed twice for 1 min with glass beads. For enzyme assays, cells were grown overnight in 50 ml YEP with various carbon sources (Table 3), harvested and resuspended in 1.5 ml buffer (0.1 M K 2 H P 0 4 pH 7.4, 1% Triton X-100, 2 mM EDTA, 2 mM dithiothreitol, 4 mM phenylmethanesulfonyl fluoride) and vortexed as above. The extracts were clarified by centrifugation at 8000 g for 10 min. Protein concentrations were determined according to Bradford [41]. Other techniques

SDS/PAGE, staining of gels and blotting of proteins onto nitrocellulose were performed according to published procedures [42, 431. Bound antisera were detected by using goat anti-(rabbit IgG) alkaline-phosphatase-conjugated antibodies and a color-developing reagent according to the instructions of the manufacturer (Promega). Plating and screening of the 3,gtll library and phages has been described previously [33].

The yeast strains used in this study are listed in Table 1 . Escherirliia coli Y1090 [lacU169 proA lon araDl39 strA supF Isolation of the pyruvate dehydrogenase complex trp22: :TnlO (pMC9)] [33] was used as the host for the Agtll library. Plasmids were amplified in E. coli JM103 [(lac-proAB) Freshly grown baker’s yeast (500 g, a gift from Gist-brothi supE44 (F‘ tra36 p r o A B f lacz)] [34]. cades b.v., Delft, The Netherlands) was resuspended in 2 1 buffer (100 mM potassium phosphate pH 7.4, 1 mM EDTA, DNA 10 pM thiamin diphosphate, 5 mM 2-mercaptoethanol, 0.4 mM phenylmethanesulfonyl fluoride, 0.5 mg/l leupeptin, The yeast DNA library in Agtll was obtained from M. 0.7 mg/l pepstatin A). After disruption of the cells by passage Snyder. Plasmids pUC4 [35] and pUC9 [36] were used for through a plunger-homogenisator (Manton-Gaulin, flow rate subcloning of fragments. Oligodeoxynucleotide mixtures de570 ml/min) and removal of cell debris by low-speed rived from N-terminal and carboxy-terminal sequences of the centrifugation, the pH was adjusted to 6.3 and poly(ethy1ene published amino acid sequences [22] of the human Elcr and glycol) 6000 added to a concentration of 7%. The suspension E I P subunits, and from a central fragment of the human was stirred for another 30 min at 3°C and the precipitate E l a subunit, were a gift from Gist-brocades b.v. (Delft, The collected by centrifugation (14000 g , 30 min). The pellet was Net herland s). Phage DNA, plasmids and yeast DNA were isolated ac- taken up in 700 ml buffer (as above, but pH 7.0) and made 2% streptomycin sulfate. Stirring was continued for 30 min. cording to standard procedures as reported previously [37, After removal of the pellet (14000 g , 25 rnin), the supernatant 381. DNA was labelled radioactively with 32Por 3 s S or nonwas incubated for 5 h at room temperature with 15 pg/ml radioactively using commercially available kits. DNAse and 15pg/ml RNAse in the presence of 10 mM MgC12. The pyruvate dehydrogenase complex was pelleted by Genetic methods ultracentrifugation for 2.5 h at 200000 x g and taken up in 12 Genetic methods have been described previously [37, 381. ml gradient buffer (100 mM Mops, pH 7.0, 1 mM EDTA, Gene disruption in the diploid strain YS60 was according to 0.2 mM dithiothreitol, 10 pM thiamin diphosphate, 0.25 mM Rothstein [39]. Separation of chromosomal DNA molecules phenylmethanesulfonyl fluoride, 0.3 mg/l leupeptin, 0.3 mg/l by orthogonal-field alternation gel electrophoresis (OFAGE) pepstatin A). Purification was concluded by centrifugation through a 15-50% sucrose gradient (60000g, 13 h). The was according to de Jonge et al. [40].

771 Table 2. Purification qf the pyruvate dehydrogenase complex from bakers' yeust One unit (U) is the amount catalyzing the production of 1 bmol NADH/min. Due to interfering activities, the pyruvate dehydrogenase in the crude samples could not be determined (n.d.) Fraction

Crude extract Poly(ethy1ene glycol) pellet Streptomycin sulfate supernatant Ultracentrifuge pellet Sucrose gradient fractions Concentrate

Volume

Protein

Activity

Specific activity

Yield

Purification

in1

mg

U

U/mg

%

-fold

2000 700 700 12 53 2

28 000 4060 2240

n.d. 972 865 300 242.8 142.8

-

-

-

0.24 0.39 2.1 23.1 23.1

100 89 31 14.7 14.7

1 1.6 8.8 97.5 91.5

1so

6.1 6.1

pooled complex-containing fractions were dialyzed against 2 mM potassium phosphate pH 7.0 and freeze-dried to a volume of 2 ml (final phosphate concentration 25 mM).

1

2

3

4

kDa

- 66

Antisera

Subunits of the complex were separated by SDSIPAGE. Unstained gels were scanned in a two-dimensional densitometer at 280 nm and the bands containing the E l a and E l j subunits cut out. The gel slices were homogenized and dialyzed against 0.9% NaCl. The suspensions were concentrated by ultrafiltration to a final subunit concentration of 100 pg protein/ml. Antibodies against the isolated subunits were obtained by immunization of rabbits with these homogenates (M. Vos and T. Hamersveld, Netherlands Cancer Institute, Amsterdam). Enzyme assays

Pyruvate dehydrogenase was assayed by the production of either NADH [44] or CoASAc from pyruvate. NADH formation was determined spectrophotometrically at 340 nm. To inhibit the oxidation of NADH by the concerted action of pyruvate decarboxylase and alcohol dehydrogenase, 150 mM pyrazole was added to the reaction. CoASAc formation was assayed by a modification of the procedure of Scislowski and Davis [45]. Since CoASAc formation was linear for more than 50 min, we improved the sensitivity of the assay by increasing the reaction time to 50 min. Furthermore, the concentration of p-dimethyl aminobenzaldehyde in the colour reaction was reduced from 6% to 1.2%. Thus it could be dissolved in ethanol instead of the toxic and volatile acetonitrile and, more importantly, the colouring reaction reached equilibrium much quicker (in less than 1 min) and disposable plastic cuvets could be used.

5

-

45

- 31

Fig. 1. SDSJPACE 0, ,>roteins. -ane 1, purified pyruvate dehydrogenase complex. The sizes of the subunits marked on the left are: E2, 56 kDa; E3, 54 kDa; X, 50 kDa; Elm, 45 kDa; EIP, 35 kDa [46]. Lane 2, Etfi subunit; lane 3, Elcr subunit. Lanes 1, 2 and 3 were stained with Coomassie brilliant blue. Lanes 4 and 5 are Western blots of cell-free extracts from strain YS62 treated with anti E1P (lane 4) or anti-Elcc serum (lane 5). The bound antisera were visualised as described in Materials and Methods.

protein degradation. The isoelectric point precipitation was omitted completely. The results of the purification are shown in Table 2. The quality of the preparation was checked by gel electrophoresis (Fig. 1, lane 1). To isolate the E l a and EIP subunits, gel slices containing these proteins were cut from denaturing SDSjPAGE gels and homogenised in buffer. The concentrated preparations were used to immunize rabbits without further purification. The resulting antisera were tested on Western blots of cell-free extracts from strain YS60. The results are shown in Fig. 1.

RESULTS

Isolation of the gene f o r the E l a subunit

Isolation ofpyruvate dehydrogenase subunits and preparation of antisera

A yeast DNA library in the expression vector l g t l l was screened with the anti-Ela serum. From a total of approximately lo5 plaques screened, four positive plaques were obtained, which were called AEla2, iEla5, IvEla10 and J"Elal1. Expression of the yeast proteins in all four isolates appeared to be independent of the lucZ promoter, since a positive signal was obtained with and without isopropyl P-Dthiogalactopyranoside induction of the lacZ promoter. Restriction analysis and hybridization studies of the DNA in the phages showed that the yeast DNA inserts of two isolates, ]"Ela2 and I E l a l 1 , were identical and both completely intern-

To obtain samples of the E l a and E l j subunits of sufficient purity to raise antibodies, we decided to isolate the entire pyruvate dehydrogenase complex first. Since published procedures were unsatisfactory in our hands, we modified the method described by Kresze and Ronft [46]. Apart from minor adaptations, like the use of 7% poly(ethy1ene glycol) instead of 3% in the first precipitation step, we removed nucleic acids by precipitation with streptomycin sulfate and reduced the incubation with DNase and RNase from 18 to 5 h to prevent

772

1 2 3 4 5 6

1 2 345 6

E X ByH ElMO I I

Ba HlBgBa X E I t

Ba

- 3.4 - 2.0

El a

E la

2.1

- 2.1

I$BFga

-1.6

El& 2/EloCl1

m Fig. 2. Restriction map of the yeast D N A inserts in iE1m phages. (A) Restriction maps. Ba = BumHI, Bg = BglII, E = EcoRI, H = HindIII, S = S o l l , X = X'hoI. The lines below the lElccll map represent the rcstriction fragments which hybridized to oligonucleotides corrcsponding to parts of the human P D H A gene described in the text. (B) Southern blot of genomic yeast DNA from strain W3031 B digested with EcoRI (lane l), BgnI (lane 2) or Hind111 (lane 3) hybridized to the non-radioactively labelled yeast DNA insert from i.Elal0

1 2 3

14

4 5

xm Il XIP X XT

TL ILm

Ix

m

PT I

Fig. 3. Chromosomal locution of yeast D N A inserts. Chromosomal D N A molecules of strain YS59 (lanes 1, 2 and 3) or strain X340215C (lanes 4 and 5) were separated by OFAGE and blotted onto nylon membranes. Parts of the blots were hybridized to various nonradioactively labelled probes. Lane 1 , yeast DNA insert from 1 E l r 5 ; lanes 2 and 4, Ty-3 DNA to visualize the bands on the blots (note that not all chromosomes contain a Ty-I element); lane 3, yeast DNA inscrt from AElalO; lane 5, yeast DNA insert from lElg21. The chromosomal assignments in the center have been taken from Carle and Olson [48]

a1 to that of a third isolate, AElalO (Fig. 2A). The insert of the fourth isolate, AElcl5, had a completely different restriction map and did not hybridize to either one of the three other inserts. Moreover, AEIa5 DNA hybridized to DNA molecules from chromosomes XI and XVI on Southern blots of genomic DNA separated by OFAGE, whereas the yeast DNA insert from /1ElalO only hybridized to chromosome V DNA (Fig. 3). Apparently, two different isolates had been obtained which both reacted strongly with the antiserum used. Only the insert of 3,Elnll, and by inference llEla2 and AElalO, however, hybridized to oligonucleotides corresponding to amino acid sequences from the N-terminal (bp 143- 163, Arg-Leu-GluGlu-Gly-Pro-Pro) or central (bp 574- 597, Glu-Asn-Asn-ArgTyr-Gly-Met-Gly) regions of the human Elcl subunit [22]. We therefore assumed that AEla2, i E l a l O and AElclll contained the gene encoding the Elcl subunit. Southern blots of genomic DNA from strain W303-1B digested with BglII, EcoRI, or

Fig. 4. lmmunohlots of extracts f r o m disruption mutunts. Cell-free extracts were prepared as described in Materials and Methods. Approximately equal amounts of protein were separated by SDSjPAGE and blotted onto nitrocellulose membranes. The blot was developed with anti-Elm serum. Lane 1, YS60-1A (haploid segregant of YS 60); lane 2, Y2a-4A; lane 3, Y2a-4B; lane 4, Y2c(-4C; lane 5, Y2m-4D; lane 6, YS62. Y2c(-4A, Y2%-4B, Y2a-4C and Y2m-4D originate from one ascus. The Ura phenotype (+ or -) of the strains is shown below the lanes

Hind111 showed that the yeast DNA insert in lElalO was either unique or a perfect repeat of more than 8 kb, the size of the single EcoRl fragment hybridizing to the yeast DNA insert from AElalO (Fig. 2B). DNA sequences showing similarity with the human PDHA gene were localized on a 1.7-kb Hind111 fragment (Wenzel and Steensma, unpublished results). To verify that this fragment indeed contained the gene for the E l a subunit, the 1.7-kb HindIII fragment on one of the chromosome V copies in the diploid strain YS60 was replaced by the yeast URA3 gene [39]. Blot hybridization of genomic DNA digested with BglII or EcoRI showed that the U R A 3 gene had integrated in the expected BglII fragment on chromosome V in each of the seven Ura' transformants tested (data not shown). Dissection of asci from these transformants resulted in tetrads containing four viable spores of which two were Ura' and two were Ura-. The spores of three complete tetrads of two different transformants were analyzed for the presence of the E l a peptide and pyruvate dehydrogenase activity. SDS/PAGE-separated cell-free extracts of YEPGal-grown cells were blotted onto nitrocellulose and incubated with anti-Ela serum. No Elcl antigenic material was detected in any of the haploid Ura' spore cultures (Fig. 4), whereas the diploid transformants and all haploid Ura- cultures showed the same 45-kDa protein as spore cultures of the parental strain YS60. The activity of the pyruvate dehydrogenase complex in these Ura strains was also strongly reduced in comparison to the Ura- strains and YS60 (Table 3). Thus gene replacement at the 1.7-kb HindIII fragment from chromosome V abolished the production of E l a peptides and simultaneously pyruvate dehydrogenase activity, showing that the gene for the E l a subunit had been disrupted. We propose to call this gene PDA I (indicating pyruvate dehydrogenase cl subunit). +

Isolation of clones using anti-EIP serum A yeast DNA library in the expression vector Agtll was screened with the anti-Elp serum. From a total of approximately 10' plaques screened, two positive phages were obtained, named lElp16 and AElP21 respectively. Like the Elcl isolates, the expression of the yeast proteins in the AElP

773 Table 3. Pyruvate dehydrogenase in cell;free extracts of wild-type and mutunt cells grown on various carhon sources One milliunit (mu) is the amount catalyzing the production of 1 nmol CoASAc/min. Cells were grown overnight in YEP with the carbon sources shown. Cell-free extracts were prepared as described in Materials and Methods. Y ~ K - ~Y2a-4B, A, Y2a-4C and Y2a-4D all originated from one ascus

A ElP21 I I

YS 60 Y 2 ~ -A4 Y2a-4B Y2a-4C Y2a-4D

Relcvant genotype

PDAIIPDAI PDAI pdal::URA3 PDAI pdal::URA3

I ‘I

i

z

3

kb

4

- 14.2 -11.7

J

-

6.5

-

2.9

- 5.0

EBa HBaHBg

El P I 6 Strain

B

EBg Ba HBaHBg E

HE

E

Specific activity when grown on -~

I% glucose

0.1% glucose

3% 2 Yo ethanol galactose

8.6 10.1 0.2

7.3 9.1 0.1 5.6 0.1

6.3 4.5 0.4 10.1 0.1

8.9 0.4

5.8 4.9 0.4 2.4 0.1

phages was independent of induction of the lacZ gene. This was confirmed when the restriction maps of the two inserts were compared. The maps overlapped partially, but were inverted with respect to the lucZ promoter in the phage DNA (Fig. 5). Southern blots of genomic DNA from strain AB972 restricted with BamHI, BglIl, EcoRI or HindIII, showed that the insert of the AE1P phages represented a unique genomic DNA fragment which was localized on chromosome XIV by OFAGE (Fig. 3). Alternatively, chromosome XIV could contain one or more repeats of the fragment. The size of the repeat would then be at least 17 kb, the sum of the three BumHI restriction fragments that hybridized to the yeast DNA insert from AElP16. The isolates hybridized neither of two oligonucleotide mixtures which corresponded to the amino-terminal (bp 118 147, Asn-Glu-Gly-Met-Asp-Glu-Glu-Leu-Glu-Arg) and carboxy-terminal (bp 877 - 900, Glu-Gly-Gly-Trp-Pro-Glu-PheGly) portions of the human E1P subunit [22]. Sequence analysis of the 1.0-kb HindIII fragment from 3.Elp21 revealed an identical sequence to the previously isolated RAP1 gene ([46], data not shown). The restriction maps of the inserts of l.Elj21 and l”ElP16were found to be similar to two of the R A P l isolates which were obtained from the same library [46]. From these results we concluded we had reisolated the R A P l gene. The position of this gene on the AElp21 insert left only 0.9 kb available for another gene. None of the open reading frames in this region, the largest of which is 250 bp, was homologous with the gene encoding the human E1P subunit (data not shown). It is therefore unlikely that the yeast gene encoding the E1P subunit would be present on the isolated clones. DISCUSSION The yeast pyruvate dehydrogenase complex is reported to contain four subunits, Ela, ElP, E2 and E3. SDSjPAGE of the purified complex indeed showed these four subunits but, in addition, we consistently observed an extra protein of approximately 50 kDa, which copurified with the complex (marked X in Fig. 1). This protein has been observed previously [47,48] and was then attributed to proteolytic products of the larger subunits or a protein kinase [47]. Since the yeast pyruvate dehydrogenase is not regulated by (de)phosphoryla-

Fig. 5. Restriction map of the yeast D N A inserts in J-EID phages. (A) Restriction maps. Symbols as in Fig. 2. (B) Autoradiograph of a Southern blot of genomic yeast DNA from strain AB972 digested with BarnHI (lane I), BgnI (lane 2), EcoRl (lane 3) or HindIII (lane 4) hybridized to the radioactively labelled yeast DNA insert from iE1P21

tion [47,48], the extra protein is probably not a protein kinase. Moreover, the concentration of the kinase, which occurs in only one to three copies in the mammalian complex, probably would have been too low for detection. Recently, a protein with a similar molecular mass has been recognized as a distinct polypeptide in mammalian pyruvate dehydrogenase complexes [SO]. It has been named component X or protein X and plays an essential role in both the binding of the E2 subunit to the complex and the activity of the complex [51]. The extra SO-kDa protein found in the yeast complex might be the yeast equivalent of protein X. The PDAI gene was isolated from a i g t l l library using antisera raised against the highly purified subunit. The resulting antiserum reduced the enzyme activity of the pyruvate dehydrogenase complex by 80 - 90% (unpublished results). The serum reacted dominantly with a protein of the expected size. Nevertheless, several other yeast proteins were bound also (Fig. 1). Hence, genes encoding minor antigenic components could have been isolated. This was indeed observed for the anti-Ela serum by which two different sequences were obtained. One was represented by the bacteriophages lwElcc2,AElalO and 1-Elall and contained the PDAl gene. The nature of the other isolate, in bacteriophage iEla5, is not known. The yeast DNA insert in this bacteriophage hybridized to two different chromosomes, XI and XVI. Recently, it has been reported that the pyruvate decarboxylase gene also hybridized two loci [15]. Pyruvate decarboxylase performs a similar reaction as the E l subunit of the pyruvate dehydrogenase complex and also requires thiamin diphosphate as a cofactor. The chromosomal locations of the two loci of the PDC gene are not known. It is unlikely, however, that the yeast DNA insert in ILEla5 contains one of the pyruvate decarboxylase genes, since its restriction map is completely different from those of P D C l , PDC3, or PDCS [12, 15, 161. To show we had indeed cloned the PDAI gene, the sequences that presumably contained all or part of the genes were disrupted by gene replacement [39]. Disruption of the PDAl gene not only abolished the production of antigenic material with the same molecular mass as the E l a subunit but, more importantly, abolished pyruvate dehydrogenase activity in haploid pdul ::U R A 3 disruption mutants. Thus we showed that the isolated sequences encoded the major antigenic component of the anti-Elcc serum and that this component formed part of the pyruvate dehydrogenase complex. From these results we concluded that we had indeed isolated the structural gene for the E l a subunit of the complex.

774 The pdul mutants were viable and did not have an apparent phenotype, although preliminary results suggest reduced growth rates on several carbon sources. Despite the absence of pyruvate dehydrogenase activity, these mutants grew on glucose media. These results corroborate physiological studies which show that, even at low glucose concentrations, part of the pyruvate is converted into CoASAc via the acetaldehyde/ acetate shunt [52]. Hence, pyruvate dehydrogenase activity is not required for cell growth. Using the anti-Elfl serum, only clones containing the RAPl gene were obtained. This result was surprising since the isolation procedure for the Elfl subunit seemed to preclude contamination with RAPl protein. Moreover, Western blots of yeast extract developed with anti-Elg serum showed no trace of the 92-kDa RAPl protein. In contrast, the antiserum used to isolate RAPI strongly reacted with a protein of approximately similar molecular mass to the E1P subunit (Fig. 2A in [46]). This protein also appears more abundant than the RAP1 protein, even after purification by DNA-affinity chromatography. Although unlikely, a serological crossreactivity between the two proteins might explain our results. Alternatively, the gene encoding the Elfl subunit could be adjacent to RAPI. This is not likely either since there is no room on the yeast DNA insert from IElfl21. There are only 0.9 kb between the end of RAPl and the end of the insert. The largest open reading frame in this area is only 250 bp. It runs towards the end of the insert and might continue in iElp16. However, it is not homologous with the cloned human gene [22, 241. Experiments are in progress to decide between these possibilities. We would like to thank M. Vos and T. Hamersveld for preparation of the antisera, M. Snyder for his generous gift of the i g t l l library and Gist-brocades n.v. for oligonucleotides. We acknowledge the help in some experiments of H. Rossdorf, J. Verdoes, T. van Wijngaarden and 0. de Vreede and thank our colleagues at the Delft University of Technology and J. A. van den Berg for helpful discussions. REFERENCES 1. De Deken, R. H. (1966) J . Gen. Microhiol. 44, 149- 156. 2. Schmitt, H. D. & Zimmermann, F. K. (1982) J . Bacteriol. 151, 1146- 1152. 3. Petrik. M., Kappeli, 0. & Fiechter, A. (1983) J . Gen. Microbiol. 129, 43-49. 4. Klppeli, 0. (1986) Adv. Microh. Physiol. 28, 181 -209. 5. Urk, H. van (1989) Ph. D. Thesis, Delft University of Technology. 6. Urk. H. van, Schipper, D., Breedveld, G. J., Mak, P. R., Scheffers, W. A. & van Dijken, J. P. (1989) Biochim. Biophys. Actu 992, 78 - 86. 7. Lim, F., Rohde, M.. Morris, C. P. &Wallace, J . C. (1987) Arch. Biochem. Biophys. 258,259 - 264. 8. Morris, C. P., Lim, F. &Wallace, J. C. (1987) Biochem. Biophys. Res. Commun. 145, 390 - 396. 9. Lim, F., Morris, C. P., Occhiodoro, F. & Wallace, J. C. (1988) J . Biol. Chem. 263, 11493 - 11497. 10. Bennetzen, J. A. & Hall, B. D. (1982)J. Biol. Chem. 257, 30183025. 1 1 . Russell, D. W., Smith, M., Williamson, V. M. & Young, E. T. (1983) J . Biol. Chem. 258, 2674-2682. 12. Schmitt, H. D., Ciriacy, M. & Zimmermann, F. K. (1983) Mol. Gen. Genet. 192, 247-252. 13. Young, E. T. & Pilgrim, D. (1985) Mol. Cell. Biol. 5, 3024-3034. 14. Kcllcrmann, E. & Hollenberg, C. P. (1988) Curr. Genet. 14, 337344. 15. Schaaff, I., Green, J. B. A,, Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, 75-81.

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