Plant Molecular Biology39: 953–967, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Heterologous expression inSaccharomyces cerevisiaeof an Arabidopsisthaliana cDNA encoding mevalonate diphosphate decarboxylase

Helene Cordier1,∗, Francis Karst2 and Thierry Berges11Laboratoire de G´enetique Physiologique et Mol´eculaire, ERS CNRS 6099, Institut de Biologie Mol´eculaire etd’Ingenierie Genetique, 40 Avenue du Recteur Pineau, 86 022 Poitiers C´edex, France (∗author for correspon-dence);2INRA, 28 rue de Herrlisheim, 68000 Colmar, France

Received 18 July 1998; accepted in revised form 19 November 1998

Key words: Arabidopsis thaliana, heterologous expression, isoprenoids, mevalonate diphosphate decarboxylase,sterols,Saccharomyces cerevisiae

Abstract

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence ofSaccha-romyces cerevisiaeidentified an EST clone corresponding to a cDNA that may encodeArabidopsis thalianaMVD(AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol andisoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA wasperformed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs.The sequence of the genomic regionof A. thalianaMVD was also obtained and Southern blot analysis on genomicDNA showed thatA. thalianacould have at least one homologous MVD gene. In order to allow heterologousexpression inS. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeastPMA1strong promoter. When expressed in yeast, theA. thalianacDNA complemented both the thermosensitiveMN19-34strain deficient in MVD, and the lethal phenotype of anERG19deleted strain. However, the wild-typesterol content was not fully restored suggesting that theA. thalianaMVD activity may not be optimal in yeast. Atwo-hybrid assay was also performed to evaluate homodimer formation of theA. thalianaMVD and heterodimerformation between the plant and yeast heterologous enzymes.

Introduction

The mevalonate pathway in eukaryotic cells leads tothe synthesis of both sterols and isoprenoids. Sterolsare mainly involved in plasma membrane structureand are precursors of steroid biosynthesis. Moreover,in yeast, ergosterol is implicated in the regulation ofthe cell cycle [17]. Isoprenoids take part in diversecellular functions, such as respiration, protein glyco-sylation and signal transduction [8]. In higher plants,this pathway leads also to a large number of plant-specific molecules including photosynthetic pigments,growth regulators (e.g. gibberellins and abscisic acid),phytotoxins, phytoalexins and other plant defence

The nucleotide sequence data reported will appear in theEMBL Nucleotide Sequence Database under the accession numbersY14325A and Y17593.

compounds. A wide variety of secondary metabolitessuch as aromatic terpenoids and natural rubbers arealso derived from this pathway [1, 5].

Mevalonate diphosphate decarboxylase (MVD;EC L-1-1-33) is a key enzyme in the isoprenoid andsterol biosynthetic pathway. It catalyses the first stepof isoprenoid biosynthesis, i.e. the decarboxylation ofthe six-carbon mevalonate diphosphate (MVA-PP) tothe five-carbon isopentenyl diphosphate (IPP). IPP isthe basic skeleton of the biosynthesis of isoprenoids.The decarboxylation catalysed by MVD is accompa-nied by the dehydratation of the substrate and thehydrolysis of one molecule of ATP and requires Mg2+[3, 11].

In plants, sterols and isoprenoid-derived moleculesare localized to various subcellular compartments inwhich they fulfil their physiological function: sterols

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are enriched in the plasma membrane, chlorophyll isexclusively found in chloroplasts, carotenoids in chro-moplasts, ubiquinone in mitochondria. The subcellularcompartmentalization of the enzymes involved in thesynthesis of these molecules has been extensively in-vestigated but remains poorly documented [1, 5, 9,12]. A novel non-mevalonate pathway for the syn-thesis of IPP has been discovered in bacteria [19]and green algae [22] and, recently, has also beendemonstrated as being the source of xantophyla andcarotenoid synthesis of higher plants [14]; these mole-cules are synthesized in the plastid compartment. Itis of interest to check the localization of the enzymesinvolved in the sterol/isoprenoid biosynthetic pathway.Therefore, isolation of a plant MVD cDNA could behelpful to characterize the precise localization of thisenzyme, and more generally to elucidate the regulationand the compartmentalization of the two pathways.

The cDNA encoding the MVD of budding yeast,human and rat have been recently isolated and char-acterized [2, 25, 26]. During these studies, severalEST clones from species such asArabidopsis thaliana,Oryza sativaandCaenorhabditis eleganswere identi-fied as potentially corresponding to cDNAs encodingMVD from these species. In order to get more infor-mation about the plant MVD and about the early stepsof isoprenoid biosynthesis in plants, we decided tosequence the full-length cDNA encodingA. thalianaMVD from the largest EST clone identified and to ex-press it in yeast under the control of an appropriatepromoter. We report here on the molecular charac-terization of theA. thaliana MVD DNA (both thecDNA and the genomic coding region) and on thefunctional complementation of two MVD-deficientSaccharomyces cerevisiaestrains with theA. thalianaenzyme.

Materials and methods

Strains and plasmids

The followingSaccharomyces cerevisiaestrains wereused: the wild-type strain FL100 (ATCC 28 383,MATa), the mutant strainMN19-34 (MAT a, erg19-34,ura3-1, trp1-1) temperature-sensitive and defective forMVD activity [4], the deleted diploid strain TB11(erg19::TRP1/ERG19, ura3-1/ura3-1, trp1-1/trp1-1)[2], and the strain L40 (MATa, trp1, leu2, his3, ade2,LYS2::lexA-HIS3, URA3::lexA-lacZ).

The Escherichia colistrains DH5α and XL1Blue(Stratagene) were used for plasmid amplification.

The Newman EST clone 123P21T7 [16] carryingthe cDNA encodingArabidopsis thalianaMVD waskindly provided by Dr Keith R. Davis from the Ara-bidopsis Biological Resource Center of Ohio StateUniversity. This clone originates from theA. thalianacDNA library built in the Escherichia coliDH10bstrain (Promega). The cDNA contained in the ESTclone 123P21T7 [16] is inserted as aSalI/NotI re-striction fragment in the polylinker of the double-strand plasmid pZL1, derived from the vectorλZipLox(Promega). pZL1 allows theα-complementation testand contains the complementary sequences for theuniversal and reverse primers.

TheE. coli replicative vectors pUC18 and pBlue-Script SK+ (Pharmacia) were used for subcloning andsequencing.

The E. coli replicative vector pGEMT-easy(Promega) that allows the direct cloning of PCR frag-ments, was used to clone the genomic MVD regionamplified by PCR. The resulting plasmids were calledpHC127AT and pHC128AT (both orientations).

The pNEV-1 [27] and pRS316 [24]S. cere-visiae/E. colishuttle vectors were used to subclone thecoding region of the cDNA encoding MVD generatedby PCR. pNEV-1 is a replicative plasmid in whicha uniqueNotI restriction site allows the insertion ofany cDNA whose transcription is under the control ofthe strong plasma membrane ATPase 1 (PMA1) pro-moter fromS. cerevisiae, thereby allowing expressionin yeast. The resulting plasmid was called pHC124AT.

The 3.7 kbHindIII fragment of pHC124AT con-taining theA. thalianaORF flanked byPMA1 pro-moter and terminator was inserted into theHindIII siteof the polylinker of the centromeric plasmid pRS316.The resulting plasmid was called pHC126AT.

The plasmids pBTM116 [28] (kindly provided byDr Jacques Camonis, INSERM U-248, Paris) con-taining thelexA-DNA binding domain and pGAD424(Clontech) containing theGAL4 activation domainwere used for two-hybrid assay. TheA. thalianacDNAamplified by PCR was cloned into theEcoRI/BamHIsites of pBTM116 and pGAD424, resulting inpLEXA-AtMVD and pGAD-AtMVD.

Preparation of plant genomic DNA

Genomic DNA was prepared fromA. thalianayoungplants (2-week old, prior to flowering) grown on solidMurashige & Skoog medium (Ducheta), according tothe Rogers and Bendich method [18], using 500 mg of

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frozen plants (all tissues). Tobacco genomic DNA waskindly provided by Dr Rémi Lemoine.

Preparation of yeast total RNA

15 OD600 units of exponentially growing yeast cellswere pelleted and resuspended in RNA extractionbuffer (100 mM LiCl, 1 mM EDTA, 100 mMTris-HCl pH 7.5). A mix of 0.5 ml PCI (phe-nol/chloroform/isoamyl alcohol 25:24:1 v/v/v), 0.5 mlRNA extraction buffer, 10µl 10% SDS and 0.5 mlof glass beads was added to the cells. Eight cycles of30 s vortexing / 30 s incubation on ice were performed,and the aqueous phase was re-extracted twice with onevolume of PCI. Aqueous phase was then adjusted with0.3 M sodium acetate and RNA were precipitated with2.5 volumes of ethanol and by storing at least 1 h at−20◦C. The RNA pellet was then resuspended in wa-ter treated with DEPC (diethyl pyrocarbonate) 1:1000v/v.

Preparation of plant total RNA

500 mg of frozen youngA. thalianaplants (2 weeksold, all tissues) grown on solid Murashige & Skoogmedium (Ducheta) were ground with liquid nitrogen,and the powder obtained was resuspended in 600µl ofREB buffer (25 mM Tris-HCl pH 8, 25 mM EDTA pH8, 75 mM NaCl, 1% SDS, 1 M 2-mercaptoethanol).Two extractions with one volume of PCI were per-formed, and the aqueous phase was washed with onevolume of CI (chloroform/isoamyl alcohol 24:1 v/v).LiCl was added to a final concentration of 2 M, andprecipitation was performed overnight at 4◦C. TheRNA pellet was then washed with 2 M LiCl and re-suspended in DEPC water. RNA was precipitated with1:10 volume of 3 M sodium acetate pH 5.2 and 2 vol-umes of ethanol for 20 min at−20◦C. The RNA pelletwas washed twice with 70% ethanol and resuspendedin DEPC-treated water.

Polymerase chain reaction

The oligonucleotides MVD3 (5′-AAAGCGGCCGCATGGCGGAGGAGAAATGG-3′) and MVD4 (5′-AAAGCGGCCGCTTATTTGGGGAGGCCAGT-3′)were used to amplify the complete open reading frameencodingA. thalianaMVD. The PCR product has asize of 1258 bp. These primers were designed ac-cording to the sequence of the whole cDNA encodingA. thaliana MVD. The sequence of MVD3 corre-sponds to the 5′ part of the cDNA and starts exactly

at the ATG codon. The sequence of MVD4 corre-sponds to the complementary sequence of the 3′ partand ends exactly at the stop codon (TAA). Both MVD3and MVD4 are flanked by aNotI linker, allowingthe subcloning of the coding region in theNotI siteof pNEV-1 cloning site. The resulting construct wascalled pHC124AT.

The oligonucleotides MVD5 (5′-ATGGCGGAGGAGAAATGGGTG-3′) and MVD6 (5′-TTATTTGGGGAGGCCAGTTTG-3′) were used to amplify the ge-nomic MVD coding region, from the start codonATG to the stop codon TAA. MVD5 starts exactly atthe ATG, and MVD6 ends exactly at the TAA. Nolinkers were added. The 2837 bp PCR product wasdirectly cloned in pGEM-T Easy (Promega), result-ing in the pHC127AT and pHC128AT constructs (bothorientations).

The oligonucleotides MVD10 (5′-AAAGAATTCATGGCGGAGGAGAAATGGG-3′) flanked by anEcoRI linker and MVD11 (5′-AAAGGATCCTTATTTGGGGAGGCCAGTTTG-3′) flanked by a BamHIlinker, were used to amplify theA. thaliana MVDcoding region from start to stop codons. The frag-ment obtained was controlled by sequencing andfused in frame with thelexA-DNA binding domain ofpBTM116 [28] and to theGAL4-activation domain ofpGAD424 (Clontech), resulting in plasmids pLEXA-AtMVD and pGAD-AtMVD used for the two-hybridassay.

The polymerase chain reaction was performed withPharmacia r-Taq DNA polymerase, under the condi-tions described by the supplier. The temperature ofannealing was 52◦C, and 30 cycles were performed.

Sequence analysis

The clone 123P21T7 is available in the EMBL data li-brary under the accession number of gb|T44736|T44736 7999. This clone was identified by homologysearch against the yeast MVD cDNA sequence, us-ing the Experimental GENINFO (R) BLAST NetworkService (NCBI).

The EST clone 123P21T7 kindly provided by DrKeith Davis was directly sequenced using universaland reverse primers. Several deletions and subcloningwere performed to obtain the complete cDNA se-quence on both strands.

The pHC127AT and pHC128AT plasmids con-taining the genomic region in both orientations, andseveral subclones, were sequenced using the univer-sal and reverse primers, allowing the obtention of the

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whole genomic region sequence, from the start codonto the stop codon.

The sequences were obtained with the dideoxynu-cleotide method [21], using the Autoread Sequencingkit with the ALF DNA sequencer (Pharmacia), andthe ABI PRISM Dye Terminator Cycle Sequencingkit with the ABI PRISM A310 sequencer (PerkinElmer). Sequences were assembled and analysed withthe DNASIS program (Hitachi). The GCG programPILEUP was used to align the translated sequencewith amino acid sequences from the SwissProt databank; the accession numbers are U49260 (humanMVD), U49261 (yeast MVD) and U53706 (rat MVD).

Southern blot analysis

10µg of A. thalianaandNicotiana tabacumgenomicDNA were digested with eitherBglII or EcoRI. TheDNA fragments were separated in a 0.8% agarosegel, blotted onto a nylon membrane (Hybond N+, A-mersham) and hybridized with a32P-dCTP-labelledprobe. The 752 bpKpnI/PstI fragment from MVDcDNA was radiolabelled by random priming. Hy-bridization was performed at 65◦C in Quick HybridBuffer (Amersham). The washes of the membranewere performed under high stringency conditions(2×5 min in 2×SSC, 0.5% SDS at room temperature,20 min in 1×SSC, 0.5% SDS at 68◦C, 3×20 min in0.1×SSC, 0.5% SDS at 68◦C).

Northern blot analysis

10 µg and 20µg of total RNA were separated ina 1.2% denaturing agarose gel containing 12.32 Mformaldehyde, and blotted onto a nylon membrane(Hybond N, Amersham). Hybridization was per-formed at 65◦C in a buffer composed of 0.25 Msodium phosphate pH 7.2, 6.6% SDS, 1 mM EDTAand 1% v/w BSA, with the 752 bpKpnI/PstI probedescribed above. Washes were performed as follows:2×5 min in 2×SSC, 0.5% SDS at room temperature,20 min in 1×SSC, 0.5% SDS at 68◦C and 2×20 minin 0.1×SSC, 0.5% SDS at 68◦C.

Reverse transcription and PCR reactions

The first strand of the cDNA synthesis was performedusing the Superscript II Reverse Transcriptase (Gibco-BRL) from 5 µg of total RNA. The synthesis of thesecond strand was performed by PCR withTaqDNApolymerase (Gibco-BRL), using the twoA. thalianaMVD-specific primers MVD5 and MVD6, with an

annealing temperature of 52◦C. The reverse tran-scription and PCR were performed according to theconditions described by the supplier.

Yeast functional complementation assays

Plasmid pHC124AT, containing the coding region ofA. thalianaMVD, was used to transform the yeast mu-tant strainMN19-34using the lithium acetate methoddescribed by Gietzet al. [7]. Transformed cells (HC0strain) were spotted on complete medium (YPD) at28◦C and 36◦C to analyse the functional complemen-tation of the plant cDNA.

The heterozygous deleted diploid strain TB11wastransformed with either pHC124AT or pHC126AT,using the same method, and transformed cells wereforced to sporulate to obtain viable transformed hap-loid cells. The remaining diploid cells were then killedby diethyl ether. Haploid transformed cells bearingboth the disrupted allele and the plasmid-borne MVD(HC1: erg19::TRP1, ura3-1, trp1-1 [pHC124AT] andHC2: erg19::TRP1, ura3-1, trp1-1 [pHC126AT])were selected on minimal medium (YNB). Haploidyof the prototrophic strains was verified by crosseswith appropriate haploid strains of known mating type.Plasmid loss was analysed on a 5-fluoro-orotic acid(5-FOA) containing medium prepared as described byGuthrieet al. [10].

Sterol level measurement

Sterols were extracted from yeast as described pre-viously by Marcireauet al. [15]. Yeast strains weregrown for 16 h in complete medium (YPD). Theamount of sterol with conjugated 5–7 double bonds(75% ergosterol and 25% ergosta-5,7-dienol) was de-termined from the maximum absorbance at 281.5 nm[23], and expressed as % of cell dry weight: % 5,7=OD281.5nm× volume of heptanolic phase (ml)× 396 /11500× dry weight (mg). The sterol composition ofeach extract was then analysed by gas chromatogra-phy, with a Carlo Erba GC 6000 chromatograph, ona SE 30 column. Cholesterol (Sigma) was used as theinternal standard to determine relative retention times(in min) as described by Bergèset al. [2].

Two-hybrid assay

The recipient strain L40 was co-transformed withboth plasmids pLEXA-AtMVD and pGAD-AtMVD,using the lithium acetate method [7]. The resulting

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strain was called T1. Strain T2 was obtained by co-transformation of strain L40 with pLEXA-AtMVDand pGAD-19wt containing theS. cerevisiaegeneERG19 encoding MVD, fused in frame with theGAL4-activation domain of pGAD424. Strain T3 wasobtained by co-transformation of L40 with pLEXA-19ts containing theerg19-34allele from the yeasttemperature-sensitive strainMN19-34, fused in framewith the lexA-DNA-binding domain of pBTM116,and pGAD-AtMVD. Strain T4 was obtained by co-transformation of L40 with pLEXA-AtMVD andpGAD-19ts containing theerg19-34allele fused inframe with GAL4-activation domain (Cordieret al.,manuscript in preparation). Co-transformed cloneswere selected on minimium medium YNB supple-mented with adenine and histidine (50µg/ml). In-dependent clones were grown on minimum mediumYNB supplemented with adenine (50µg/ml) for thedetection of the activation of theHIS3reporter gene.

Independent transformants were grown overnighton a Whatman #40 filter on complete YPD medium.Filters were immersed 10 min in liquid nitrogen andthen placed on top of a Whatman #3 filter prewet in Zbuffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mMKCl, 1 mM MgSO4, 38 mM 2-mercaptoethanol,pH 7), containing 0.2 mg/ml of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (XGal). XGal assays wereperformed by incubating the filters for 3 h at 37◦C.Blue coloration after incubation is indicative ofβ-galactosidase activity.

The transformants were grown to stationary phasein minimum medium containing 3% glucose (w/v)supplemented with adenine and histidine (50µg/ml).The β-galactosidase activity was determined fromcrude protein extracts as described previously [20].

Results

Identification of anA. thalianacDNA encoding MVD

Database searches with the amino acid sequence ofthe yeast mevalonate diphosphate decarboxylase ledto the identification of an EST that may contain thecDNA encoding theA. thalianahomologue of this en-zyme [25]. The partial nucleotide sequence availablein the data library was 471 bp long, and its deducedamino acid sequence presented 56.7% identity over 74amino acids with theS. cerevisiaeMVD amino acidsequence. Since this sequence was homologous to theN-terminal part of the yeast MVD, the 123P21T7 EST

clone was expected to contain the complete ORF. Thisclone was provided by Dr Keith R. Davis from theArabidopsis Biological Resource Center of the OhioState University for further analysis.

Analysis of the cDNA and the deduced amino acidsequence

We first established a restriction map of theNotI/SalIinsert contained in 123P21T7 by digestion with sev-eral restriction enzymes (Figure 1). The estimated sizeof theNotI/SalI insert was 1800 bp. A nucleotide se-quence of 1695 bp was obtained, containing a 1239 bplong open reading frame (AtMVD1 cDNA, EMBLaccession number Y14325A). The 3′ end of the in-sert including the poly(A) region was not sequenced.The ATG start codon is located at position 118 rela-tive to the first nucleotide of the insert, and the TAAstop codon at position 1354. The open reading framepotentially encodes a protein of 412 residues with apredicted molecular mass of 45 585 Da and a theoricalisoelectric point of 6.33. A putative polyadenylationsignal AATAAA is found at position 1634 in the3′-untranslated region.

The predicted amino acids sequence ofA. thalianaMVD was aligned with the various MVD sequencesavailable: rat MVD (accession number U53706),human MVD (U49260) andS. cerevisiaeMVD(U49261), and the results are summarized in Figure 2.The protein presents 57%, 56% and 54% strict iden-tity, and 69%, 72% and 71% similarity with yeast,human and rat MVDs, respectively. TheA. thalianaMVD is clearly homologous to the other eukary-otic MVDs previously described, especially in itsN-terminus half.

Analysis of theA. thalianaMVD genomic region

The genomic region corresponding to the MVD cod-ing region was obtained by PCR amplification fromA. thalianagenomic DNA, using the primers MVD5and MVD6 that were designed to target the regionlocated between the start codon and the stop codon.The entire sequence of the 2837 bp genomic re-gion that was isolated was subsequently identified(AtMVD1 gene, EMBL accession number Y17593).Nine exons and eight introns were identified by se-quence comparison with the cDNA. The localiza-tion and sequence of the 5′ and 3′ intron splicesites are reported in Table 1. The splice sites werecompared to theArabidopsisintron splice-site table

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Figure 1. Organization ofArabidopsis thalianaMVD cDNA and genomic region, and Southern blot analysis. a. Map of the whole 1800 bpMVD cDNA containing a 1239 bp ORF. b.KpnI/PstI (752 bp) probe used for Southern analysis. It overlaps a 2114 bp region on the genomicDNA. c. Schematic drawing of the genomic region. The region from the start to the stop codon is 2837 bp long and contains nine exons (E1 toE9) and eight introns (I1 to I8). d. Southern blot analysis under high-stringency conditions. Lane 1,A. thalianagenomic DNA digested withBglII (10 µg); lane 2,A. thalianagenomic DNA digested withEcoRI (10µg); lane 3, tobacco genomic DNA digested withBglII (10 µg); lane4, tobacco genomic DNA, digested withEcoRI (10µg).

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Figure 2. Alignment of the amino acid sequence ofArabidopsis thalianamevalonate diphosphate decarboxylase with the proteins encoded byrat, human and yeast genes. The alignment was performed using the PILEUP GCG program. The accession numbers ofA. thaliana, rat, humanand yeast MVDs are Y14325A, U53706, U49260 and U49261 respectively.

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Table 1. Organization of the genomic region ofA. thalianaMVD. The genomic part corresponding tothe translated region is composed of nine exons and eight introns. The positions were determined fromthe predicted ATG start codon. 5′ and 3′ intron splice-sites are indicated for each intron.

Exons Size Position Introns Size Position 5′ splice 3′ splice

(bp) (bp) site site

E1 210 1− 210 I1 156 211− 366 AAG ! GTA TAG ! GAA

E2 199 367− 565 I2 455 566− 1020 TAG ! GTA CAG ! TTT

E3 64 1021− 1084 I3 232 1085− 1316 AAG ! GTA TAG ! GCA

E4 145 1317− 1461 I4 309 1462− 1770 GTG ! GTA CAG ! GTT

E5 87 1771− 1857 I5 155 1858− 2012 AAG ! GTA TAG ! GAA

E6 158 2013− 2170 I6 99 2171− 2269 CAG ! GTA CAG ! GAT

E7 49 2270− 2318 I7 112 2319− 2430 GAG ! GTA CAG ! ATT

E8 125 2431− 2555 I8 80 2556− 2635 GAG ! GTG TAG ! TTA

E9 202 2636− 2837

available in theA. thaliananucleotide sequence data-base (http://genome-www.stanford.edu/Arabidopsis)and were found to match perfectly with the consensussequences. A map of the organization of the exons andintrons is presented in Figure 1.

Southern blot analysis

The 752 bp KpnI/PstI probe prepared from theA. thalianaMVD cDNA overlaps a 2114 bp region onthe genomic part including exons 2 to 8 and introns 2to 7 (Figure 1). The genomic DNA was digested withtwo restriction enzymes,BglII or EcoRI. It is impor-tant to notice that the washing steps were done underhigh-stringency conditions (Materials and methods).

ThreeBglII fragments were expected to hybridizethe probe, since there are twoBglII sites in the re-gion covered by this probe. The size of one of thesefragments should be 831 bp. The size of the twoother fragments could not be determined, since theuntranslated regions upstream from the start codonand downstream from the stop codon have not beenisolated.

Only one fragment was expected to be detected fortheEcoRI digestion, since there is noEcoRI site in theregion overlapped by the probe. The size of thisEcoRIfragment should be higher than 2837 bp, but could notbe precisely determined for the same reasons.

The results of the Southern analysis are presentedin Figure 1. FourBglII fragments hybridized to theprobe, with estimated sizes of 2700 bp, 1250 bp,850 bp and 760 bp respectively, and twoEcoRIfragments, which estimated sizes were 8400 bp and4050 bp. The number of bands obtained in compar-

ison to the number of expected fragments for theAtMVD1 gene suggests the probable existence of asecond gene. Given the high-stringency conditionsused for the washing steps (see Materials and meth-ods), the degree of homology between the two genesshould be high.

On the other hand, no hybridization could be de-tected on tobacco genomic DNA (Figure 1), evenwhen lower-stringency conditions were used (notshown). This may be due to the larger size of thetobacco genome relative toA. thaliana.

Analysis of the MVD expression inA. thaliana

Expression of MVD in the plant was studied by north-ern blot, with the same MVD-specific probe as forthe Southern analysis. Two different amounts (10 and20µg respectively) of total RNA extracted from wholeyoung plants were loaded. The results are shown inFigure 3, and reveal the presence of a very faint bandof the expected size. The signal could only be detectedusing 20µg of total RNA. This result could be ex-plained by a low expression of MVD mRNA in thewhole plant, or a tissue-specific expression.

In order to increase the detection level of the MVDRNA, a reverse transcription reaction followed byPCR withA. thalianaMVD-specific primers MVD5and MVD6 (Materials and methods) was performedon A. thaliana total RNA. The results presented inFigure 3 show that a single specific fragment of theexpected size (1240 bp) was amplified, confirming thenorthern blot result.

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Figure 3. Expression ofArabidopsis thalianaMVD in plant and heterologous expression in yeast. a. Expression ofArabidopsis thalianaMVDstudied by northern blot analysis, using the 752 bpKpnI/PstI MVD-specific probe and hybridized to 10µg (lane 1) and 20µg (lane 2) ofA. thalianaRNA prepared from whole 2-week old plants (Materials and methods), and 10µg (lane 3) and 20µg (lane 4) of total RNA of ayeast strain deleted for MVD and bearing a single copy of the plant MVD cDNA (HC2:erg19::TRP1 [pHC126AT]). b. Reverse-transcriptionPCR (RT-PCR) withA. thalianaMVD-specific primers (MVD5 and MVD6). The reaction was performed on 5µg of plant total RNA (lane1) and 1µg of yeast total RNA (HC2, lane 2). 123P21T7 DNA was added as a positive control and size indicator (lane 3); a negative controlwithout DNA was also included (lane 4).

Heterologous expression in yeast of the plant cDNA

In order to further characterize the putativeA. thalianaMVD, we decided to check whether the plant enzymeis functional in yeast, by assaying its ability to comple-ment two yeast strains deficient in MVD. One of thesetwo strains is the temperature-sensitive strainMN19-34 [4]. It harbours a recessive mutation that replacesLeu-79 by a Pro in the coding sequence of theERG19gene [2]. Due to the leaky character of the defectiveallele, when the strain is grown at permissive tem-perature, the sterol biosynthetic pathway produces theminimal ergosterol amount required for growth (ca.0.2% of dry weight) and no intermediates of the path-way can be detected ([2], and Table 2). The secondstrain bears a deleted copy of theERG19gene, andtherefore cannot survive without any plasmid-borneMVD [2].

We first constructed the pHC124AT plasmid con-taining the whole coding region ofA. thalianaMVD,including its own start and stop codons, cloned underthe control of the promoter and terminator of the yeastplasma membrane ATPase gene (PMA1) [27]. ThePMA1 promoter is a strong promoter and, moreover,the vector used is a high-copy-number plasmid. Thus,this construction should lead to the overproduction ofthe mRNA encoding theA. thalianaMVD. To achievethis construct, the coding sequence of theA. thalianaMVD was amplified by polymerase chain reaction

(PCR) from 123P21T7, using the MVD3 and MVD4oligonucleotides that were designed according to thecDNA sequence, and cloned into the pNEV-1 vector togenerate the pHC124AT plasmid. The sequence of theamplified product was verified and remains identicalto the cDNA sequence.

This construct was used to transform the yeasttemperature-sensitive strain defective in MVD activ-ity (MN19-34 strain). The transformants were firstselected at permissive temperature and then replica-plated at 36◦C. All the Ura+ transformants couldclearly grow at non-permissive temperature, althoughslower than the wild type, as shown in Figure 4,whereas the parental mutant strain (MN19-34) couldnot grow at this temperature.MN19-34harbouring thepHC124AT plasmid was called HC0.

In order to express the plant cDNA in a yeaststrain deleted for MVD, we decided to transformwith pHC124AT a yeast diploid strain in which acopy of the functionalERG19gene is disrupted bythe TRP1 marker (TB11 strain). We then analysedthe haploid progeny of these transformed cells aftersporulation. Haploid strains bearing both the yeast dis-rupted gene copy and the plant cDNA (Ura+, Trp+)were obtained (HC1). Surprinsingly we found thatthey were indistinguishable from the wild-type strain,whatever the growth conditions (Figure 4), in contrastto HC0. No plasmid loss was observed after five daysof subculturing on complete medium. Moreover, the

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Figure 4. Yeast complementation with the plant cDNA encoding MVD. Growth of the yeast mutant strainMN19-34defective in MVD activity,complemented with the plant cDNA (HC0 strain:MN19-34[pHC124AT], lane 3) was compared toMN19-34transformed with the pNEV-1plasmid (lane 4, negative control), and to the wild-type FL100 (lane 2). The growth of the haploid disrupted strain complemented with the plantcDNA (HC1: erg19::TRP1 [pHC124AT], lane 1) was also assayed. The strains were grown on complete medium at 28◦C (a) and 36◦C (b),using three dilutions of yeast cell suspensions (1, 10, 100).

Figure 5. Analysis of the resistance to 5-fluoro-orotate(5-FOA). Resistance to 5-FOA of several yeast strains (lane A,erg19::TRP1[pPA295-2], lane B,erg19::TRP1/ERG19[pPA295-2],lane C, HC1: erg19::TRP1[pHC124AT], lane D,erg19::TRP1/ERG19[pHC124AT]) was assayed on minimal mediumsupplemented with uracil and tryptophan, using three dilutions ofyeast cell suspensions (1, 10, 100). The plasmid pPA295-2 bearsthe yeast functional MVDERG19 gene, and theURA3 gene as aselective marker [2]. The haploid and diploid yeast strains bearingthe disrupted MVD gene copy withTRP1 marker and plasmidpPA295-2 (lanes A and B) were included as negative and positivecontrols, respectively. Haploid strains which are not able to losetheir plasmid do not grow on 5-FOA (lanes A and C). Diploidstrains able to lose their plasmid can grow on such a medium (lanesB and D).

haploid disrupted strain transformed with pHC124AT(HC1) could not grow on a 5-fluoro-orotate-containingmedium (Figure 5), confirming that this strain was notable to lose its plasmid.

The experiment was repeated, but with pHC126AT,a centromeric (monocopy) plasmid containing the

whole MVD coding region under the control of thePMA1 promoter. The results were identical (data notshown): haploid strains bearing both the yeast deletedcopy and the centromeric plasmid with the plantcDNA (HC2) were viable, with a wild-type phenotype,and could not lose their plasmid.

Therefore, we concluded that the cDNA used inthis study complements both the yeast mutant defec-tive allele of theMN19-34strain and the disruptedMVD gene copy of the TB11 strain, confirming thatit encodes theA. thalianaMVD.

A northern blot analysis was performed on totalRNA of yeast complemented with a single copy of theplant cDNA (HC2 strain:erg19::TRP1[pHC126AT]).Total RNA (10 and 20µg) was hybridized with theA. thalianaMVD-specific probe presented in Figure 1.The results are shown in Figure 3. For 10µg of totalyeast RNA, a single signal with the expected size wasobserved, the intensity of which is much higher thanthe one observed for the plant total RNA. RT-PCRon the complemented yeast total RNA gave a singlespecific amplified fragment of the expected size (seeFigure 3). These results confirmed that the plant cDNAwas efficiently expressed in yeast when controlled bythe strongPMA1promoter.

Sterol analysis

Considering the slow growth rate of the thermosen-sitive strain complemented with theAtMVD1 cDNA(HC0: MN19-34 [pHC124AT]), we decided to mea-sure the amount of sterol in a disrupted back-

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Table 2. Quantitative and qualitative analysis of the sterol composition.

Strain % of 5,7- Sterol composition (%)

dienol

Zymosterol Ergosterol Fecosterol Ergosta-

5,7-dienol

FL100 0.84± 0.06 15 55 5 25

MN19-34 0.15± 0.01 0 92 0 8

HC0 0.18± 0.03 ND ND ND ND

HC1 0.54± 0.03 11 69 3 17

HC2 0.41± 0.02 ND ND ND ND

ND, not determined.The amount of sterol was measured in the haploid strains bearing the disrupted MVDgene copy and complemented with the plant cDNA, HC1 (erg19::TRP1[pHC124AT],high-copy-number plasmid) and HC2 (erg19::TRP1[pHC126AT], centromeric plas-mid), in comparison to the thermosensitive strainMN19-34defective for MVD activity,MN19-34-complemented strain (HC0:MN19-34[pHC124AT]) and the wild-type strainFL100. The strains were grown on complete medium at 28◦C, and the amount of sterolis expressed in mg per 100 mg dry weight. The HC0 strain was grown on minimummedium supplemented with tryptophan. The results are the average of four independentexperiments for each strain. The sterol composition of these strains was analysed by gaschromatography (see Materials and methods).

ground. Two independent haploid strains, bearing thedeleted MVD allele and complemented with the plantMVD cDNA, carried either on a high copy num-ber plasmid (strain HC1:erg19::TRP1[pHC124AT])or a centromeric plasmid (strain HC2:erg19::TRP1[pHC126AT]), were used, and the relative sterolcomposition was analysed by gas chromatographyand compared to theMN19-34temperature-sensitivestrain, to the HC0 strain and to the wild-type parentalstrain. For sterol extraction, all these strains weregrown at 28◦C in YPD complete medium, exceptHC0, grown in selective minimal medium (YNB tryp-tophan) for plasmid maintenance. The results areshown in Table 2. While a typical sterol contentof the wild-type strain (FL100) grown in completemedium was about 0.8 mg per 100 mg of dry weight,the amount obtained with HC1 (ca. 0.5–0.6 mg per100 mg) and HC2 (0.4 mg per 100 mg) did not reachthis level. The amount of sterol synthesized increaseswith the number of copies of the plant cDNA: thisamount is significantly higher than the sterol contentof the mutantMN19-34(ca. 0.15 mg per 100 mg) andthe HC0 strain (ca. 0.18 mg per 100 mg) but remainslower than in the wild-type strain. GC analysis showedthat the plant cDNA restores a sterol biosynthesis qual-itatively comparable to the wild-type strain, since thevarious intermediates of the pathway can be detectedand their respective quantities differ only slightly fromthe wild-type strain (Table 2).

Two-hybrid assay

In order to evaluate the putative homodimer forma-tion of A. thaliana MVD a two-hybrid assay wasperformed. TheAtMVD1 coding sequence was am-plified by PCR and fused in frame to the DNA-binding domain oflexA (pLEXA-AtMVD), and tothe GAL4-activation domain (pGAD-AtMVD). Co-transformants of the L40 recipient strain bearing bothplasmids were selected. Transcription of the two re-porter genes was then assayed. When theHIS3 re-porter gene is switched on, the strain can grow onsynthetic medium lacking histidine (Figure 6a). Tran-scription of thelacZ reporter gene was evaluated bothby an XGal assay (Figure 6b) and by measuringβ-galactosidase specific activity (Table 3). The resultsshow that theA. thalianaMVD forms homodimersinvivo (T1 strain), as well as the yeast MVD (Cordieret al., manuscript in preparation). Heterodimer for-mation between the plant and the wild-type yeastenzymes (T2 strain) was also detected by these as-says. By contrast, heterodimer formation could not bedetected when the plant enzyme was used in combina-tion with the mutated yeast MVD (Figure 6, Table 3,strains T3 and T4).

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Figure 6. Two-hybrid assay. a. Co-transformed strains T1 to T4 (see Materials and methods) were grown on minimum medium supplementedwith adenine and histidine (50µg/ml) and on minimum medium supplemented with adenine (50µg/ml), using three dilutions of cell suspensions(1, 10, 100), for the analysis of the activation of theHIS3 reporter gene. b. XGal assay was performed to analyse the activation of thelacZreporter gene with strains T1 to T4. The blue coloration (dark grey on picture) is indicative ofβ-galactosidase activity.

Table 3. Measurement ofβ-galactosidase activity.

Strain T1 T2 T3 T4 L40

Specific activity 0.314± 0.144 0.259± 0.104 0.060± 0.034 0.058± 0.019 0.048± 0.012

β-galactosidase activity expressed in nmol ofo-naphthol per min per mg was measured from protein crudeextracts from strains T1 to T4 (see Materials and methods), grown in minimum medium containing 3% (w/v)glucose supplemented with adenine and histidine (50µg/ml). L40 was included as a negative control. Theresults are the average of duplicate assays with two independent transformants.

Discussion

With the recent finding of a novel mevalonate-independent pathway in various organisms, it remainsto be determined whether these two routes take placein different and totally independent compartments inplants. Isolation and characterization of the cDNAsencoding the enzymes involved in the isoprenoid path-way could allow the study of the heterologous expres-sion of the protein of interest, its purification and thepreparation of specific antibodies, in order to elucidatethe subcellular localization of the enzymes.

In the mevalonate pathway, IPP is produced bythe decarboxylation of MVA-PP. This catalytic step isperformed by the mevalonate diphosphate decarboxy-lase. Based on the study of Toth and Huwyler [25,26], who identified human, rat and yeast MVDs, wedecided to characterise the cDNA encoding the MVD

from A. thaliana, identified by sequence comparisonwith databases. Comparison of the sequence of thiscDNA with the previously described MVDs revealedthat it contains the whole open reading frame encodingthis enzyme. The predicted coding sequence displaysan overall identity of about 55% with the other eu-karyotic MVDs. The best matches were obtained inthe N-terminal part of the protein. This cDNA proba-bly encodes a cytoplasmic protein, since no additionaltransit peptide is predicted at the N-terminus of theprotein.

To further characterize theAtMVD1 gene, thegenomic coding region was isolated by PCR fromthe start to the stop codons. The PCR product is2837 bp long, and is split into nine exons and eightintrons, with 5′ and 3′ splice sites perfectly matchingto A. thalianaconsensus intron splice-sites. A South-ern blot analysis suggests the existence of a second

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gene encoding MVD in the genome ofA. thaliana,since both digestions used in this study revealed thepresence of additional bands which were not expectedaccording to the restriction map of theAtMVD1 cod-ing region. These bands could have been due to apartial digestion of the genomic DNA, but this hypoth-esis was ruled out, since it was not compatible withthe restriction maps. Our data suggest a high degree ofconservation between theAtMVD1 gene and its puta-tive homologue since the additional hybridized frag-ments were observed when high stringency washeswere used. The existence of a second MVD gene isconsistent with the study of otherA. thalianagenes in-volved in isoprenoid biosynthesis. Indeed at least twoother enzymes involved in the mevalonate pathway areencoded by two independent genes [6, 13]. In the caseof farnesyl diphosphate synthase (FPS), one of the twogenes encodes a mitochondrial enzyme [6].

In order to confirm the identity of the proteinencoded by this cDNA, its heterologous expressionwas performed in yeast. To achieve a high level ofexpression, theA. thaliana MVD coding sequencewas cloned under the control of a strong yeast pro-moter, the promoter of the plasma membrane ATPase(PMA1) gene, and was introduced into a high-copy-number plasmid. This construct was transformed intoa leaky yeast mutant defective for the mevalonatediphosphate decarboxylase (MN19-34strain) [4]. Thistemperature-sensitive strain bears a single-point muta-tion in theERG19gene encoding the yeast MVD [2].At the permissive temperature, the ergosterol biosyn-thetic pathway is functional, but produces very lowamounts of 5,7-dienol, i.e. 0.15–0.2% of dry weight,as compared to ca. 0.8–0.9% for a wild-type strain.When introduced into this strain, theA. thalianaMVDcDNA clearly suppressed the temperature-sensitivephenotype. However, growth of the complementedstrain (HC0) was still significantly impaired in com-parison to the wild type, and the amount of sterol pro-duced was not significantly different from the amountof sterol synthesized in the mutantMN19-34.In orderto further characterize the level of complementationof an MVD-defective yeast and to standardize thegrowth conditions of the various strains studied, wedecided to express the heterologous MVD cDNA inanERG19-disrupted background.

The erg19::TRP1/ERG19 (TB11) strain wastransformed with pHC124AT and haploid progenyharbouring both the null-ERG19 copy and theA. thaliana MVD cDNA was obtained (HC1:erg19::TRP1[pHC124AT]), demonstrating that this

enzyme is indeed functional in yeast. In addition, thecomplemented strain could not afford plasmid losswhen plated on 5-fluoro-orotate. Moreover, these hap-loid complemented strains (HC1) grew perfectly well,by contrast to the slow growth of HC0 (MN19-34[pHC124AT]).

The sterol content of these strains was studiedand compared to both the wild-type and theMN19-34 temperature-sensitive strain grown in the samemedium (YPD). HC1 produces ca. 0.55% of dryweight, whereasMN19-34produces only ca. 0.15%and HC0 less than 0.2%. Nevertheless the sterol leveldoes not reach that of the wild-type strain (ca. 0.84%).When we further characterized the sterol compositionby GC analysis, we observed that the sterol biosyn-thetic pathway is functional, since the various inter-mediates are detected in the complemented disruptedstrain (HC1), as well as in the wild type. Indeed, inMN19-34, almost only ergosterol is detected show-ing that the pathway is able to produce the minimalamount of ergosterol necessary for growth. The onlynoticeable difference in HC1 versus the wild type re-sides in the ergosta 5,7-dienol/ergosterol ratio: in thewild type, this ratio is higher (about 0.45) than in HC1(0.24), while it is only about 0.09 in theMN19-34strain.

The amount of sterol produced in HC1 is lowerthan in the wild-type FL100 (Table 1). This couldbe due to a lower specific activity of theA. thalianaMVD. However, it is important to notice that we ob-served a similar result when the yeastERG19gene wasoverexpressed [2]. Therefore, it was not excluded thatthis lower amount of sterol could be the result of afeedback regulation of the sterol biosynthetic pathwaywhen MVD is overproduced. In order to check thishypothesis, the plant MVD cDNA was cloned into acentromeric plasmid, under the control of the samePMA1 promoter. Theerg19::TRP1/ERG19(TB11)strain was transformed with this construct, and thehaploid complemented strain (HC2) showed the samephenotype as HC1. The amount of sterol in HC2 waslower than in HC1 (0.41% versus 0.54%). By con-trast, when TB11 was complemented with a singlecopy of the yeast functional allele, it produces 0.8%of sterol as the wild type [2]. In our case, decreasingthe number of copies of the plant cDNA reduces thesynthesis of sterols. Therefore, the amount of sterolin yeast strains complemented with the plant MVDcDNA (HC1 and HC2) seems to be the consequenceof a lower specific activity of the plant enzyme in yeast

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rather than the effect of a feedback regulation due toits overproduction.

The situation observed with the defectiveMN19-34mutant strain is intriguing, since the complementationwith the plant cDNA is only partial (HC0), whereasit is almost total for the disrupted strains (HC1 andHC2). The partial complementation phenotype ob-served in HC0 is well correlated with an apparent lackof increase in the production of sterols. To explainthis phenotype, two hypotheses could be proposed.The defective allele ofMN19-34may titrate the sub-strate, thereby reducing its accessibility to the plantMVD. Another possibility is that theerg19-34alleleproduces defective subunits that could interact withthe plant MVD, thereby altering the enzymatic ac-tivity in the HC0 strain. In agreement with the latterhypothesis, MVD of chicken and rat liver [3, 26]and of man (M. Toth, personal communication) havebeen reported to be homodimeric enzymes. Using atwo-hybrid assay, we recently showed that theSac-charomyces cerevisiaeMVD also forms homodimerswhereas the mutated enzyme produced by theMN19-34 temperature-sensitive strain is severely impaired indimerization at permissive temperature (Cordieret al.,manuscript in preparation). Using the same method,we obtain here evidence for homodimer formation ofA. thalianaMVD subunits. Moreover, we show thata monomer of the plant enzyme can interact with thewild-type yeast MVD, confirming the high degree ofconservation amongst MVD of these species. How-ever we could not detect any interaction between themutated yeast MVD and theA. thaliana monomer.Therefore, the partial complementation in HC0 cannotbe explained by the formation of defective plant-yeastheterodimers.

Expression ofAtMVD1 was studied inA. thalianaand in the yeast strain HC2 by northern blot analysisand RT-PCR. Expression in plant was shown to below, since a weak signal was observed, and only for20µg of total RNA. Nevertheless, we cannot exclude atissue-specific expression that would lead to a dilutionof the MVD specific mRNA when using total RNA.By contrast, the plant cDNA was efficiently expressedin yeast, since a strong signal was observed with only10µg of total HC2 RNA.

The results reported here which describe the mole-cular characterization of a plant MVD represent apreliminary step to study the compartmentalizationand regulation of the mevalonate pathway in higherplants.

Acknowledgements

We thank Dr Keith R. Davis and the ArabidopsisBiological Center of the Ohio State University for pro-viding the EST clone 123P21T7, Dr Rémi Lemoinefor helpful advice for northern blot analysis, and DrMatthieu Régnacq for critical reading of the manu-script. This work was supported by grant 9/95 fromGREG/INRA. H.C. was recipient of grant 95040 fromthe French Ministère de l’Enseignement Supérieur etde la Recherche.

References

1. Bach TJ: Some new aspects of isoprenoids biosynthesis inplants: a review. Lipids 30: 191–202 (1995).

2. Bergès T, Guyonnet D, Karst F: Characterisation of a Leu-to-Pro mutation in a conserved sequence of mevalonate diphos-phate decarboxylase in yeast. J Bact 179: 4664–4670 (1997).

3. Cardemil E, Jabalquinto AM: The mechanism of action ofmevalonate-5-pyrophosphate decarboxylase. Trends BiochemSci 8: 7 (1983).

4. Chambon C, Ladevèze V, Blanchard L, Javelot C, VladescuB, Karst F: Sterol pathway in yeast. Identification and prop-erties of mutant strains defective in mevalonate diphosphatedecarboxylase and farnesyl diphosphate synthetase. Lipids 26:633–636 (1991).

5. Chappell J: Biochemistry and molecular biology of the iso-prenoid biosynthetic pathway in plants. Annu Rev PlantPhysiol Plant Mol Biol 46: 521–547 (1995).

6. Cunillera N, Arro M, Delourme D, Karst F, Boronat A, FerrerA: Arabidopsis thalianacontains two differentially expressedfarnesyl-diphosphate synthase genes. J Biol Chem 271: 7774–7780 (1996).

7. Gietz RD, St Jean A, Woods RA, Schiestl RH: Improvedmethod for high efficiency transformation of intact yeast cells.Nucl Acids Res 20: 1425 (1992).

8. Goldstein JL, Brown MS: Regulation of mevalonate pathway.Nature 343: 425–430 (1990).

9. Goodwin TW, Mercer EI: The control of lipid metabolism. In:Grant JK (ed), pp. 37–40. Academic Press, London (1963).

10. Guthrie C, Fink GR: Guide to yeast genetics and molecularbiology. Meth Enzymol 194: 310–311 (1991).

11. Jabalquinto AM, Alvear M, Cardemil E: Physiological as-pects and mechanism of action of mevalonate 5-diphosphatedecarboxylase. Comp Biochem Physiol 90B: 671–677 (1988).

12. Kreuz K, Kleinig H: Synthesis of prenyllipids in cells ofspinach leaf, compartmentation of enzymes for formation ofisopentenyl diphosphate. Eur J Biochem 141: 531–535 (1984).

13. Kribii R, Arro M, Del Arco A, Gonzales V, Balcells L,Delourme D, Ferrer A, Karst F, Boronat A: Cloning and char-acterisation of theArabidopsis thaliana SQS1 gene encodingsqualene synthase. Involvement of the C-terminal region ofthe enzyme in the channeling of squalene through the sterolpathway. Eur J Biochem 249: 61–69 (1997).

14. Lichtenthaler HK, Schwender J, Disch A, Rohmer M: Biosyn-thesis of isoprenoids in higher plant chloroplasts proceeds viaa mevalonate-independent pathway. FEBS Lett 400: 271–274(1997).

967

15. Marcireau C, Guyonnet D, Karst F: Construction and growthproperties of a yeast strain defective in sterol 14 reductase.Curr Genet 22: 267–272 (1992).

16. Newman T, De Bruijn FJ, Green P, Keegstra K, KendeH, McIntosh L, Ohlrogge J, Raikhel N, Somerville S,Thomashaw M, Retzel E, Somerville C: Genes galore: a sum-mary of methods for accessing results from large-scale partialsequencing of anonymousArabidopsiscDNA clones. PlantPhysiol 106: 1241–1255 (1994).

17. Rodriguez RJ, Taylor FR, Parks LW: A requirement forergosterol to permit growth of yeast sterol auxotrophs oncholestanol. Biochem Biophys Res Comm 106: 435–441(1982).

18. Rogers SO, Bendich AJ: Extraction of milligram amounts offresh, herbarium and mummified plant tissues. Plant Mol Biol5: 69–76 (1985).

19. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H: Iso-prenoid biosynthesis in bacteria: a novel pathway for theearly steps leading to isopentenyl diphosphate. Biochem J 295:517–524 (1993).

20. Rose MD, Winston F, Hieter P: Methods in Yeast Genetics:A Laboratory Course Manual. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY (1990).

21. Sanger F, Nicklen S, Coulson AR: DNA sequencing withchain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467 (1977).

22. Schwender J, Seemann M, Lichtenthaler HK, Rohmer M:Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyru-vate/glyceraldehyde 3-phosphate non-mevalonate pathway inthe green algaScenedesmus obliquus.Biochem J 316: 73–80(1996).

23. Servouse M, Karst F: Regulation of ergosterol biosynthesis inSaccharomyces cerevisiae.Biochem J 240: 541–547 (1986).

24. Sikorski RS, Hieter P: A system of shuttle vectors and yeasthost strains designed for efficient manipulation of DNA inSaccharomyces cerevisiae.Genetics 122: 19–27 (1989).

25. Toth MJ, Huwyler L: Molecular cloning and expression of thecDNAs encoding human and yeast mevalonate pyrophosphatedecarboxylase. J Biol Chem 271: 7895–7898 (1996).

26. Toth MJ, Huwyler L, Park J: Purification of rat liver meval-onate pyrophosphate decarboxylase. Prep Biochem Biotech-nol 26: 47–51 (1996).

27. Villalba JM, Palmgreen MG, Berberian GE, Fergusson C,Serrano R: Functional expression of plant plasma membraneH+-ATPase in yeast endoplasmic reticulum. J Biol Chem 267:12341–12349 (1991).

28. Vojtek AB, Hollenberg SM, Cooper JA: Mammalian Ras in-teracts directly with the serine/threonine kinase Raf. Cell 74:205–214 (1993).