Brassicaceae Express Multiple Isoforms of Biotin Carboxyl Carrier Protein in a Tissue-Specific

Brassicaceae Express Multiple Isoforms of Biotin CarboxylCarrier Protein in a Tissue-Specific Manner1

Jay J. Thelen*, Sergei Mekhedov, and John B. Ohlrogge

Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits.One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single genein Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identityto AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms wereexpressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted withpurified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea(Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylatedpolypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 proteinwas present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarilyexpressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcriptreflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereasAtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected indeveloping seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereasdeveloping seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate thatoilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upongenome complexity.

The committed step for de novo fatty acid biosyn-thesis is the formation of malonyl-coenzyme A (CoA),catalyzed by acetyl-CoA carboxylase (ACCase, EC6.4.1.2). ACCase catalyzes the ATP-dependent carbox-ylation of acetyl-CoA through a carboxylation andcarboxyltransferase two-step reaction (Guchait et al.,1974). The number and organization of ACCase iso-forms is variable in plants (Sasaki et al., 1995). Allplants contain a homomeric cytosolic isoform that isproposed to be involved in flavonoid and long-chainfatty acid biosynthesis (Shorrosh et al., 1994). A secondisoform is present within the plastid stroma of plantcells and is involved in de novo fatty acid biosynthesis(Kannangara and Stumpf, 1972; Alban et al., 1994). Ingraminae plants, plastidial ACCase is a homomericenzyme, whereas in dicots and non-graminae mono-cots it is composed of at least four polypeptides as-sembled as a complex of approximately 600 kD (Sasakiet al., 1995; Ohlrogge and Jaworski, 1997). Because denovo fatty acid biosynthesis occurs predominantly inplastids (Ohlrogge et al., 1979) and malonyl-CoA isimpermeable to the plastid envelope, it is widely ac-cepted that plastidial ACCase is the key initial enzymein this pathway. In addition to a heteromeric isoform,plastids from Brassicaceae plants also contain a homo-

meric ACCase, the role of which remains unknown(Schulte et al., 1997).

A wealth of direct and indirect evidence suggestsplastidial ACCase is one major control point for light-induced fatty acid biosynthesis in leaves and likelyone rate-limiting step for this pathway in other or-gans. Evidence in support of this premise is summa-rized below. Measurement of in vivo acyl-acyl carrierprotein (ACP) pools from isolated pea (Pisum sati-vum) chloroplasts indicated malonyl-ACP wasshifted to acetyl-ACP after the transition to darkness(Post-Beittenmiller et al., 1991, 1992). Biochemicalproperties of plastidial ACCase indicate optimal ac-tivity during light-adapted stromal conditions (Niko-lau and Hawke, 1984; Hunter and Ohlrogge, 1998).Regulation of ACCase via reversible redox activation(Sasaki et al., 1997) or reversible protein phosphory-lation (Savage and Ohlrogge, 1999) is mediated bylight. In graminae monocots, inhibitor studies cou-pled with in vivo labeling indicated strong flux con-trol coefficients at this step (Page et al., 1994). Insuspension cells, feedback inhibition of fatty acidbiosynthesis occurred at the ACCase step (Shintaniand Ohlrogge, 1995). Targeting of a homomericACCase to the plastids of rapeseed resulted in a 5%increase in total seed fatty acid content (Roesler et al.,1997).

Besides being present in dicotyledon plant andgreen algal plastids, multisubunit ACCases are alsofound in gram-negative and gram-positive pro-karyotes (Li and Cronan, 1992a, 1992b; Marini et al.,1995). Multisubunit ACCases are comprised of biotin

1 This work was supported by the National Science Foundation(grant no. MCB94 – 06466) and by the Michigan Agricultural Ex-periment Station.

* Corresponding author; e-mail [email protected]; fax517–353–1926.

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carboxyl carrier protein (BCCP), biotin carboxylase(BC), a-carboxyltransferase, and b-carboxyltransferasesubunits. Though the organization of the plant com-plex is unknown, the prokaryotic counterpart is ahomodimer of BC polypeptides assembled with a ho-modimer of BCCP, which is loosely associated to aheterotetramer of a- and b-carboxyltransferase sub-units (Guchait et al., 1974). In higher plants theb-carboxyltransferase subunit is plastid-encoded (Liand Cronan, 1992c; Sasaki et al., 1993), whereas theremaining three subunits are nuclear-encoded. Genesor cDNAs for the nuclear-encoded subunits have beencharacterized from Arabidopsis (Choi et al., 1995; Baoet al., 1997; Sun et al., 1997), oilseed rape (Brassicanapus; Elborough et al., 1996), soybean (Reverdatto etal., 1999), and pea (Shorrosh et al., 1996). The BCCPsubunit from Arabidopsis was previously reported tobe a single gene copy (CAC1, Choi et al., 1995). Incontrast, the allotetraploid relative oilseed rape con-tains at least six BCCP gene copies (Elborough et al.,1996). The oilseed rape isoforms group into two dis-tinct classes based upon amino acid and nucleotidesequence comparisons. The previously identified Ara-bidopsis BCCP (AtBCCP1; accession no. AF236873) ismost similar to class one oilseed rape BCCP subunits.In this report we present evidence for a second BCCPisoform from Arabidopsis, which is a homolog to classtwo BCCPs from oilseed rape. Based upon semiquan-titative immunoblot analyses we conclude that classone BCCPs are strongly expressed in most plant or-gans, whereas class two BCCPs accumulate in devel-oping seed and might have an evolved role in fattyacid biosynthesis for lipid deposition.

RESULTS

In a search of the Arabidopsis expressed sequencetag (EST) database using the AtBCCP1 polypeptideas a search query, a total of 13 putative BCCP ESTswere found. Two of these 13 were partial cDNAs thatcorrespond to the previously described AtBCCP1transcripts. The 11 remaining cDNAs encoded a pro-tein related to AtBCCP1, primarily in the C-terminalone-half of the polypeptide. Eight of these 11 en-coded the entire open reading frame (ORF) and fouralso contained the entire 59-untranslated region(UTR; GenBank accession nos. H37386, H37396,AI992947, and N38652). cDNA clone H37386 wascompletely sequenced and is reported here asAtBCCP2. The deduced polypeptide exhibits 42%amino acid identity with AtBCCP1, 40% with oilseedrape class one isoforms, 75% with oilseed rape classtwo isoforms, and 50% with soybean BCCP. The genefor AtBCCP2 (accession no. AF223948) was recentlysequenced and mapped to chromosome V. Basedupon the intron/exon structure of these two genes(Fig. 1B) and their location on chromosome V it ap-pears the second gene arose from an ancestral geneduplication event.

The AtBCCP2 cDNA is 1,235 bp in length with a765-bp ORF starting with an initiating codon at bp 94and a stop codon at bp 859. A consensus translationstart motif (90aacaatggc) containing the initiating Metis present at the 59 end and a poly(A) initiation signal(1190aaataaa) at the 39 end. The ORF encodes a 255-amino acid polypeptide with a predicted Mr of27,248. The highest similarity between AtBCCP1 andAtBCCP2 is concentrated at the C terminus, in theproximity of the biotinylation motif 217EAMKLM-NEIE226, which contains the putative biotinyl Lys(Samols et al., 1998; Fig. 1). The amino terminus(approximately 50 amino acids) resembles a plastid-targeting peptide as revealed by organellar sortingalgorithms. This putative plastid targeting peptide isonly 30% identical to AtBCCP1 and soybean BCCP-targeting peptides, but is 85% identical to those inclass two oilseed rape BCCP isoforms. The low sim-ilarity to soybean BCCP for which information aboutplastid processing is known (Reverdatto et al., 1999)precludes an accurate prediction for AtBCCP2.Therefore, to verify this protein is plastidial and es-timate the apparent Mr of the mature protein, in vitrotranslated protein was incubated with intact peachloroplasts and was assayed for protein uptake.

In vitro translation of AtBCCP2 transcript resultedin a 34-kD radiolabeled protein, approximately 7 kDlarger than the predicted molecular mass (Fig. 2).When incubated with intact pea chloroplasts, radio-labeled AtBCCP2 was imported with greater than50% efficiency. Chloroplast import was verified byprocessing of the precursor protein to a 25-kD maturepolypeptide and protection of this protein to exoge-nous thermolysin protease. As a control for proteaseactivity, detergent was added to permeabolize theenvelope, which rendered the radiolabeled proteinsusceptible to proteolysis. These data demonstratedthat the cDNA-encoded protein contained a transitpeptide of approximately 9 kD, which was capable ofdirecting it to plastids.

Recombinant Expression of BC and BCCPs andReconstitution of BC-BCCP Half-Reaction of ACCase

To confirm the molecular masses of AtBCCP1 and 2and to verify these were biotin-containing proteins,the coding regions for the predicted mature proteinswere PCR-amplified and subcloned into expressionvectors. The N-terminal amino acids of mature AtB-CCP1 and 2 were predicted to be Ser64 and Thr56,respectively, based upon loose conservation with theexperimentally determined processing site for soy-bean BCCP (Fig. 1). Two AtBCCP2 fusion proteinswere expressed in Escherichia coli. Fusion one con-tained a (His)6-tag within a 41-residue amino-terminalfusion to the mature AtBCCP2 polypeptide and fusiontwo contained only a MG-dipeptide at the amino-terminus to initiate translation for the matureAtBCCP2 polypeptide. Protein expression in the

Biotin Carboxyl Carrier Protein Isoform Two from Arabidopsis

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protease-deficient E. coli host BL21(DE3) resulted inhighly induced polypeptides of 32 and 22 kD for thetwo respective constructs, as observed by Coomassie

Blue staining and immunoblot analyses with anti-biotin antibodies. For comparison, when fused to a38-residue tag, AtBCCP1 migrated as a 42-kD

Figure 1. Amino acid sequence comparison of plant and cyanobacterial BCCPs. Alignment was performed with Clustal Xsoftware using default parameters. Shading reflects the degree of amino acid conservation; black shading indicates aminoacid identity with all polypeptides. Plastid processing site for soybean BCCP occurs between residues 48 and 49. Bycomparison, the mature AtBCCP2 was postulated to start with Thr-56, indicated by an asterisk. GenBank accession numbersare X90730, oilseed rape4; U23155, Arabidopsis1; U40666, soybean; L14863, Anabaena; D64001, Synechocys; andU38804, P. purpurea. B, Gene organization of Arabidopsis BCCP isoforms. A scale model of intron and exon (white boxes)organization for Arabidopsis BCCP isoforms one and two are illustrated. Scale bar at the lower right is 0.1 kb in length.GenBank accession numbers are as follows: At1, AB005242 and At2, AP002074.

Thelen et al.

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polypeptide during SDS-PAGE (Fig. 3), 10 kD largerthan a similar fusion for AtBCCP2. Because the size ofAtBCCP2 polypeptide without a (His)6-tag did notagree with the chloroplast imported mature AtBCCP2,it was reasoned that the predicted mature start site(Thr56) was invalid. We inevitably conclude that re-combinant expressed AtBCCP2 was not complete ma-ture protein. When expressed under standard growthconditions, detection of recombinant AtBCCP2 withanti-biotin antibodies was poor, an indication of insuf-ficient biotinylation. It was previously noted that re-combinant expression of the dihydrolipoamide acetyl-

transferase component to the mitochondrial pyruvatedehydrogenase complex required exogenous lipoicacid for complete lipoylation of apoprotein (Thelen etal., 1999). It was, therefore, hypothesized that aug-menting AtBCCP expression cultures with free biotinmight ameliorate the low biotinylation frequency. Theaddition of biotin during growth and inductionphases significantly improved the biotinylation effi-ciency, which demonstrated the E. coli biotin ligasewas capable of biotinylating this plant-derived BCCP(Fig. 3B).

Mature AtBC (starting with Cys71), expressed as a36-amino acid fusion, migrated as a 55-kD polypep-tide, which is close to the 51 kD size of native Ara-bidopsis BC. Recombinant (His)6-tagged AtBCCPsand BC were purified to greater than 90% homoge-neity by Ni-chelate chromatography (data notshown). The yield of rAtBC was 10 mg L21 cultureand subsequent to purification was concentrated to37 mg mL21 without loss of solubility. From threepreparations the yield of rAtBCCP1 was 1.6, 4.4, and3.8 mg L21, whereas rAtBCCP2 was 7.2, 22, and 24mg L21 culture media. Purified rAtBC had a specificactivity of 0.64 6 0.04 (sd) nmol CO2 min21 mg21

protein when assayed under the conditions describedhere. This value for purified enzyme is modest whencompared with the specific activities observed fromlysed pea chloroplasts (0.2–0.7 nmol min21 mg21)assayed under the same conditions. The low valuefor rAtBC is possibly due to the amino-terminal tagpresent on the recombinant form and the lack ofBCCP partner protein, which together might hindercorrect folding. Because activity was completelyabolished by boiling rAtBC prior to assaying, this isclearly the result of an enzymatic activity. As shownin Table I, both rAtBCCP isoforms were capable of

Figure 3. Expression of Arabidopsis BCCP isoforms in E. coli. A and B, Recombinant BL21(DE3) cells were grown for 6 hin Luria broth (plus antibiotic) at 37°C with shaking. Cells were aliquoted (0.2 mL) into three culture tubes containing 3 mLof Luria broth and nothing (control), 2 mM isopropylthio-b-galactoside, or 2 mM isopropylthio-b-galactoside plus 0.1 mM

biotin and grown for another 6 h. Cells were then collected by centrifugation, resuspended in 0.4 mL of SDS-PAGE samplebuffer, and heated at 95°C for 15 min prior to SDS-PAGE. A, Coomassie Blue-stained gel containing 5 mL of poly-(His)AtBCCP2-induced cell lysates. B, Anti-biotin stained immunoblot of A containing 0.5 mL of each sample. C, Anti-biotinstained immunoblot of recombinant AtBCCP2, minus fusion tag (approximately 5 mg of cell lysate; lane 1), poly-(His)AtBCCP2 (50 ng of purified protein; lane 3); and poly-(His) AtBCCP1 (approximately 5 mg of cell lysate; lane 4) resolved nextto Arabidopsis total silique protein (10 mg) for comparison.

Figure 2. Chloroplast import of Arabidopsis BCCP2. Intact pea chlo-roplasts were incubated with in vitro translated AtBCCP2 lysate (lane1) for protein uptake. The chloroplast suspension was then aliquoted,washed, and re-isolated (lane 2), or treated with thermolysin proteaseafter import (lane 3) or protease treated in the presence of TritonX-100 detergent (lane 4) prior to resolving by SDS-PAGE. AtBCCP2precursor (P) and mature (M) polypeptides were visualized by auto-radiography. Mr markers are indicated.




accepting 14CO2 from rAtBC, confirming their activ-ity as cofactors. Free biotin at the same concentrationgave a 23- to 100-fold lower rate. When specific ac-tivities are compared, rAtBCCP2 appears to be thesuperior carboxy acceptor.

Differential Expression of Arabidopsis BCCP Isoforms

The presence of two BCCP genes in Arabidopsismight be an indication of expression or biochemicaldifferences that are important for ACCase function.To determine the expression patterns for these iso-forms, northern-blot analyses were performed. Us-ing a probe containing the 39 one-half of AtBCCP1cDNA, transcript abundance from various organswas analyzed. Though amino acid identity betweenthe two BCCP isoforms is high near the carboxylterminus, the nucleotide identity is sufficiently lowto prevent cross-hybridization under the conditionsemployed here (Fig. 4). The transcript for AtBCCP1was detected in all organs analyzed, which includedroots, leaves, flowers, and siliques. AtBCCP1 tran-script abundance was similar in 14-d leaves to de-veloping siliques and was estimated to be 0.001% to0.005% of the total RNA, based upon standard DNAcontrols. Estimating the rRNA to be in 100-foldexcess over mRNA, AtBCCP1 transcript representedabout 0.1% to 0.5% of mRNA in developing leavesand siliques. In agreement with Choi et al. (1995)we observed that AtBCCP1 transcript is down-regulated during leaf development since 14-d ro-sette leaves contain approximately 3-fold more tran-script than 40-d rosette leaves. Although developingleaves and siliques contained the highest amount ofBCCP1 transcript, flowers were also rich in thistranscript, containing 92% as much. Of all the or-gans analyzed, roots contained the least amount oftranscript, 58% as much as developing leaves.

An early indication of AtBCCP2 transcript expres-sion came from microarray analyses using cDNAsderived from a developing seed EST library (Girke etal., 2000). Comparing total mRNA derived from de-veloping seeds and leaves the ratio of expressionusing four different AtBCCP2 ESTs averaged 5.1.These data indicated developing seeds contained

several-fold more transcript than developing leaves.Northern analyses showed AtBCCP2 transcript wasmostly expressed in flowers and developing siliquesand minimally expressed in roots and leaves. Theoverall abundance of AtBCCP2 transcript was ap-proximately 4-fold lower than AtBCCP1, when spe-cific activity of the hybridization probes and expo-sure times were considered. Taken together, theexpression profiles indicate isoform one is present inall organs and more abundant overall compared withisoform two.

To confirm differential expression of these BCCPisoforms, protein levels were monitored with anti-biotin antibodies. Anti-biotin antibodies were usedinstead of isoform-specific antibodies so the abun-dance of each isoform could be directly compared.Arabidopsis organs display predominantly three bi-otinylated polypeptides, 25 6 3 (sd), 35 6 3, and 88 62 kD in size. The 88-kD polypeptide was previouslycharacterized as the 3-methylcrotonoyl-CoA carbox-

Figure 4. Transcript expression analysis of Arabidopsis BCCP iso-forms. Total RNA (30 mg) isolated from total roots, 14-d rosetteleaves, 40-d rosette leaves, flowers, and developing siliques wasanalyzed. As a loading control, 16 S rRNA amounts are shown. DNAprobes were quality-controlled and assayed for cross-hybridizationusing 15 ng of double-stranded DNA (dsDNA). Cross-hybridizationbetween the two isoforms was negligible. Intensity and area of thehybridization signals were quantitated with Image Quant softwareand expressed as relative percentages.

Table I. Activity assays of purified recombinant BC and BCCP isoformsBiotin carboxylase activity was quantitated as the amount of 14C transferred from NaH14CO3 to free biotin as determined by stability to

bubbling with unlabeled CO2. Biotin carboxylase assay was performed with 50 mM d-biotin, 72 mg of purified rAtBC, and 5 mM NaH14CO3

(approximately 3,000 dpm nmol21). Reconstitution assay of purified recombinant BC and BCCP isoforms was performed with equimolar amountsof BC and BCCP (or free biotin as a substitute for BCCP) as indicated below. All values are the mean of at least three samples and SD is notedin parentheses.

Biotin Carboxylase Assay BC-BCCP Reconstitution Assay

Sample Specific activity Sample Specific activity

pmol CO2 min21 mg21 pmol CO2 min21 mg21

Minus rAtBC (background) 12 (1.7) 4.2 mM biotin 0.0 (0.0)Boiled rAtBC 14 (1.3) 22 mM biotin 0.2 (0.05)rAtBC 640 (4.0) 22 mM rAtBCCP1 4.5 (1.0)

22 mM rAtBCCP2 21.0 (10)

Thelen et al.



ylase (Song et al., 1994) and the 35-kD polypeptide, aputative BCCP to ACCase (Choi et al., 1995). The25-kD biotin-containing protein has never been re-ported from Arabidopsis, but has an apparent Mridentical to the mature size of chloroplast importedAtBCCP2 (Fig. 2) and more similar to recombinantAtBCCP2 than AtBCCP1 (Fig. 3C).

The expression pattern for the 35-kD polypeptideresembles the profile for AtBCCP1 transcript; mostabundant in 14-d leaves, flowers, and developing sil-iques, but lower in roots and 40-d leaves (Fig. 5A). Ofall the organs analyzed, 8 d-after-flowering seeds con-tained the greatest amount of 35-kD BCCP, approxi-mately 4-fold more than in 14-d leaves. Using purifiedrecombinant AtBCCPs as standards, the 35-kD BCCPwas estimated to be approximately 0.05% of the totalprotein in 14-d leaves, flowers, and siliques. The 25-kD

polypeptide is expressed predominantly in siliques,though a faint band was observed in flowers (Fig. 5, Aand B). Closer inspection of dissected siliques revealedthe 25-kD polypeptide is not present in the siliquepod, but rather specifically expressed in the develop-ing seeds (Fig. 5C). Throughout Arabidopsis seed de-velopment, the 35-kD biotin-containing polypeptidewas generally 2-fold more abundant than the 25-kDform (Fig. 6A). Both polypeptides were temporallyregulated during seed development and expression ofAtBCCP2 was highest in 7- to 10-d-after-floweringseeds and was difficult to detect during earlier stages.

In the related oilseed plant rapeseed, four BCCPpolypeptides were observed in developing seeds, 22,25, 35, and 37 kD in size (Fig. 6B). The 35-kD BCCPwas also present in developing leaves, however, the22-, 25-, and 37-kD BCCPs were not observed indeveloping leaves from equally loaded gels. Expres-sion of these three BCCPs peaked around 21 d afterflowering and unlike developing seeds from Arabi-dopsis, the 25-kD BCCP was nearly equal in abun-dance to the 35- and 37-kD forms during seeddevelopment.

Ion-Exchange Chromatography of ArabidopsisSilique Extracts

It was previously observed that pea chloroplast BCand BCCP remain associated during salt elution fromdiethylaminoethyl- (DEAE) sepharose chromatogra-phy, whereas the carboxyltransferase subunits easilydissociate from the BC-BCCP partner proteins (Shor-rosh et al., 1996). To determine if the two biotinylatedpolypeptides from Arabidopsis seeds were assem-bled with BC, a total protein extract from developingsiliques was applied to a DEAE-sepharose matrix,washed with a low ionic strength wash buffer, andwas subsequently eluted with a step gradient of in-creasing sodium chloride in wash buffer (Fig. 7).Approximately 80% of BC and BCCPs from the sol-uble extract bound to the DEAE matrix as deter-mined by immunoblot analysis. A single Arabidopsispolypeptide of 51 kD cross-reacted with anti-castorBC antibodies, which is similar in size to variousother plant BCs (Roesler et al., 1996). Most of the51-kD polypeptide eluted between 80 and 140 mmsodium chloride, which coincided with the elution ofthe two biotin-containing polypeptides. Co-elutionwith BC is supportive evidence that both of thesebiotinylated proteins are assembled with BC and rep-resent the two BCCP isoforms.

DISCUSSION

Our interest in control of fatty acid biosynthesis inoilseeds has led to the investigation of the multisub-unit plastidial ACCase and its regulation at the bio-chemical and molecular levels. With the completionof the Arabidopsis genome, the genes that comprise a

Figure 5. Protein expression analysis of Arabidopsis BCCP isoforms.BCCPs were detected with anti-biotin, alkaline phosphatase-conjugated antibodies. Relative molecular weights for BCCPs are35 6 3 (SD) and 25 6 3 kD for isoforms one and two, respectively. A,Total Arabidopsis protein (10 mg) was extracted from roots, 14-drosette leaves, 40-d rosette leaves, flowers, developing siliques, anddeveloping seeds 8 d after flowering for immunoblot analyses. Ar-rows indicate BCCP isoforms. B, Higher amount (40 mg) of root and40-d leaf protein was immunoblotted to determine if AtBCCP2 waspresent in these organs. Only the 35-kD form (BCCP1) was detectedin these organs. C, Immunoblot analysis of total Arabidopsis protein(20 mg) from closed or open flowers, developing siliques, developingsilique pods, and developing seeds (mixed stages). Amount of eachBCCP polypeptide was quantitated with Image Quant software and isexpressed as relative percentages of the maximum value. Ratios ofBCCP levels are also noted.




multisubunit plastidial ACCase have all been identi-fied. The BC and a- carboxyltransferase subunits areapparently encoded by a single nuclear gene,whereas b-carboxyltransferase is encoded by a singleplastome gene (Mekhedov et al., 2000). It was sug-gested that the BCCP subunit was also encoded by asingle nuclear gene based upon a Southern blotprobed with AtBCCP1 (Choi et al., 1995). In thisreport we characterize the presence of a second geneby identifying a cDNA encoding a second isoform forthis ACCase subunit.

Even though the amino acid identity is only 42% tothe previously characterized isoform, the evidencesummarized below conclusively demonstrates thecDNA described in this report encodes a secondBCCP isoform to Arabidopsis plastid ACCase. Thehallmark BCCP biotinylation motif is present in thededuced amino acid sequence (Fig. 1). In vitro trans-lated protein is efficiently imported and processed inchloroplasts (Fig. 2). Recombinant protein is biotin-ylated and is an active acceptor of carboxy groupsfrom BC (Fig. 3; Table I). A biotin-containing proteinof a similar molecular mass is present in Arabidopsisseeds and co-elutes with BC during ion-exchangechromatography (Figs. 5–7).

The reason two BCCP isoforms are present in Ara-bidopsis is not clear, though it is intriguing since theother ACCase subunits are encoded by single genes.To help understand this, the expression patterns ofthese isoforms was investigated. An earlier investi-gation showed that AtBCCP1 transcript is highly ex-pressed in developing leaves (Choi et al., 1995). Herewe show this transcript is also present in roots, and ishighly expressed in flowers and siliques. This tran-script expression profile is similar to that observedfor AtBC transcript that was quantitated by northern-blot and promoter-b-glucuronidase analysis (Bao etal., 1997). In that investigation siliques were the pri-mary organ where b-glucuronidase protein accumu-lated, containing 25% more activity than flowers. It

has been observed previously that in oilseed plants,developing leaves and seeds are two organs thatexhibit high rates of fatty acid biosynthesis (Ohlroggeand Jaworski, 1997). As expected, developing leaveswere rich in AtBCCP1 transcript, accumulating toapproximately 0.1% of mRNA. Flowers and siliquescontained similar amounts of transcript, which prob-ably reflects the demand for lipids during pollinationand reserve deposition (Evans et al., 1992). It is strik-ing that mature leaves are nearly devoid of this tran-script, suggesting flux through ACCase, and conse-quently fatty acid biosynthesis, is minimal at thispoint in development. In an alternate manner, thismight indicate a low rate of protein turnover. How-ever, since BCCP protein was abundant in 14-dleaves yet barely detectable in 40-d leaves, the pro-tein must be degraded during leaf development.

Unlike AtBCCP1, AtBCCP2 transcript was notabundant in roots or developing leaves, instead flow-ers and siliques contained most of the message, albeitat approximately 4-fold lower amounts than

Figure 6. Expression of BCCP isoforms during Arabidopsis and rapeseed development. BCCP polypeptides were detectedwith anti-biotin, alkaline phosphatase-conjugated antibodies. Arrows indicate the major biotinylated polypeptides. A,Arabidopsis developing seed harvested 4 to 12 d after flowering (5 mg of protein) was resolved next to 14-d developingleaves (15 mg) for comparison. Top and bottom biotinylated polypeptides correspond to BCCP1 and 2, respectively. BCCPswere quantitated and expressed as relative percentages. Ratio of absolute BCCP levels are also noted. B, Developingrapeseed harvested 1.5 to 6.0 weeks after flowering (100 mg) was resolved next to developing leaf protein (100 mg).

Figure 7. Association of Arabidopsis BC-BCCP polypeptides by co-elution from DEAE-sepharose. Arabidopsis developing silique pro-teins were applied to an ion-exchange matrix, washed with fivecolumn volumes of low ionic strength wash buffer, and eluted witha sodium chloride step gradient of 1.0-mL fractions. Equal volumes ofeach fraction were resolved by SDS-PAGE for immunoblotting withanti-castor BC or anti-biotin antibodies.

Thelen et al.



AtBCCP1. These data were in agreement with mi-croarray analyses, revealing lower expression in de-veloping leaves compared with developing seeds(Girke et al., 2000). Based upon EST abundance itmight be argued that AtBCCP2 is more abundantthan AtBCCP1. In general, this is an approximatemethod to compare transcript abundance (Mekhedovet al., 2000); cDNA construction and library amplifi-cation steps can alter the representation of transcriptsfrom RNA preparations. Because eight out of 11AtBCCP2 ESTs contain the entire ORF, whereas thetwo AtBCCP1 ESTs contain less than 50% of the ORF,perhaps the AtBCCP1 transcript has secondary struc-ture hindering reverse transcription. This is onepossible explanation for the under-representation ofAtBCCP1 transcript in Arabidopsis cDNA libraries.A different library of 10,500 ESTs derived from Ara-bidopsis developing seed mRNA contained eightAtBCCP2 versus three AtBCCP1 cDNAs (White et al.,2000). The abundance of AtBCCP2 ESTs from thislibrary correlates with our prediction (based upontranscript abundance), but AtBCCP1 EST abundanceis approximately 10-fold lower.

Analysis of these isoforms at the protein level re-quired evidence for a difference in apparent Mr. Thelow sequence homology between the two Arabidop-sis isoforms in conjunction with their low Mr led tothe speculation that these isoforms could be resolvedby conventional SDS-PAGE. We predicted thatAtBCCP2 would have a lower apparent mass thanAtBCCP1 since it contained fewer amino acids and alower percentage of Pro and Ala residues thanAtBCCP1 in the hinge region upstream of the bioti-nylation domain. It was necessary to confirm thisexperimentally, since it is well documented that theseproteins migrate anomalously during SDS-PAGE (Liand Cronan, 1992a) and processing site predictionswere based upon a poorly conserved homolog. Invitro synthesized AtBCCP2 migrated at an apparentmolecular mass of 34 kD, and after chloroplast im-port was processed to 25 kD. Both of these masses arehigher than those predicted for the precursor andmature polypeptides, which is attributed to the hy-pervariable hinge domain (Li and Cronan, 1992a).The hinge region is rich in Pro and Ala residues toincrease the flexibility for active-site coupling (Brock-lehurst and Perham, 1993). Pro- and Ala-rich regionsalter the migration of proteins during SDS-PAGE,resulting in a larger apparent size. Anomalous migra-tion during SDS-PAGE has also been observed withdihydrolipoamide acetyltransferases, which also con-tain Pro, Ala-rich flexible-hinge domains necessary foractive-site coupling (Thelen et al., 1999). Since recom-binant AtBCCP2 without a poly-(His) fusion migratedat a molecular mass of 22 kD, the predicted start site ofThr56 must be incorrect. Based upon the 32-kD appar-ent size of the 41-amino acid (His)6-tag fusion, matureAtBCCP2 likely begins near Val42.

There are six presently known biotinylated en-zymes in plants, namely homomeric ACCase (plas-tidial and cytosolic), 3-methylcrotonoyl-CoA car-boxylase, propionyl-CoA carboxylase, pyruvatecarboxylase, and BCCPs. In addition, a biotin-containing protein of unknown function is abun-dant in pea seeds (Duval et al., 1994). Of these, onlyBCCPs are low molecular mass proteins that areabundant in developing tissues. Based upon thesecriteria, the biotinylated polypeptides of 25 and 35kD are likely BCCP subunits. The 25-kD isoform isidentical in size to chloroplast-imported AtBCCP2and similar to recombinantly expressed protein. The35-kD isoform is most likely AtBCCP1 since recom-binant protein for this isoform is 10 kD larger thanAtBCCP2 (Fig. 3). Further support of this nomencla-ture is observed in the expression patterns for theseisoforms and their relative abundance. AtBCCP1transcript is observed in all organs, much like the35-kD polypeptide. AtBCCP2 transcript is primarilypresent in reproductive organs, in agreement withthe 25-kD isoform. The transcript for isoform two isapproximately 4-fold less abundant than isoformone, which is roughly true for the 25-kD protein incomparison with the 35-kD protein. Although it ispossible the smaller isoform is actually a proteolyticderivative of the larger, more abundant form weargue against this for two reasons. The protein ex-tracts in this investigation were prepared under de-naturing conditions. The 25-kD form is only ob-served in flowers and seeds, whereas the 35-kDform is present in all organs.

The presence of two BCCP genes suggests at leasttwo different types of ACCase complexes exist inArabidopsis plastids. Although the significance ofthis is unclear, some possibilities are discussed here.Expression analyses indicate AtBCCP1 could be con-sidered a housekeeping isoform since it is present inall Arabidopsis organs. In agreement with this pro-posed function, down-regulation of AtBCCP1through antisense technology resulted in chlorotic,curled, and variegated leaves, demonstrating theconstitutively expressed isoform is critical for main-tenance of cellular lipids (Fatland et al., 1999).AtBCCP2, on the other hand, appears to have a morespecific role since it is predominantly expressed inreproductive organs. Expression patterns such asthese have been observed for other multigenic fami-lies of fatty acid biosynthetic proteins and one par-ticularly well-characterized gene family is the ACP.At least three Arabidopsis ACPs are constitutivelyexpressed, whereas three more appear to be specificto seed, leaf, or root organs (Hlousek-Radojcic et al.,1992). This level of complexity within the simpleArabidopsis genome was enigmatic for a number ofyears.

It was recently discovered that optimal desaturaseactivity for the production of unusual monoenic fattyacids required a seed-specific coriander ACP (Suh et




al., 1999). Similar ACPs from spinach and E. coli didnot support the high rates observed in coriander seedextracts. Furthermore, mitochondrial and Cuphea lan-ceolata ACPs promote short-chain termination whencompared with a plastidial and a related C. lanceolataACP isoform, respectively (Shintani and Ohlrogge,1994; Schutt et al., 1998). The reason for this differ-ence is uncertain, but indicates ACPs may have spe-cific interactions with desaturase and perhaps thio-esterase function and have co-evolved with unusualfatty acid synthesis in oilseeds. Like ACPs, BCCPshave no catalytic activity, but instead function morelike cofactors since free biotin can substitute forBCCP with E. coli and plastidial ACCases (Guchait etal., 1974; Sun et al., 1997). However, the catalyticefficiency (Vmax/Km ratio) of biotin versus BCCP is8,000- and 2,000-fold lower in carboxylase and car-boxyltransferase assays, respectively (Blanchard etal., 1999). Activity differences between free andBCCP-conjugated biotin indicate association of theBCCP apoprotein with biotin carboxylase and car-boxyltransferase subunits is critical for optimalACCase activity. In an alternate manner, orderedpresentation or increased solubility of the biotin co-factor through a biotin-protein conjugate might alsoexplain these activity differences. Further evidencefor the involvement of apo-BCCP in active-site cou-pling is observed here. Both purified rAtBCCP iso-forms were preferred to free biotin as carboxy accep-tors from rAtBC (Table I). Although rAtBCCP2appears to be a superior carboxy-acceptor than rAt-BCCP1, this might be explained by differences inbiotinylation or native conformation and further ex-perimentation will be necessary to determine whichisoform is optimal for ACCase activity. These find-ings further define the role of BCCP in ACCase func-tion and suggests that amino acid differences be-tween the Arabidopsis BCCPs, in combination withdifferential expression profiles, might be one mech-anism for regulating ACCase activity.

The possibility that BCCP isoforms might influenceoverall ACCase activity led to the speculation thatthese genes have diverged to perform specific roles inoilseed plants. If so, conserved homologs should existfor the two Arabidopsis BCCPs. Dendrogram analysiswith all known BCCPs revealed at least two differentsubclasses within the plant kingdom (Fig. 8). TheBCCPs from Arabidopsis are split between these twophylogenetic classes as follows. AtBCCP1 and oilseedrape isoforms 1 and 2 define group one, whereasAtBCCP2 and oilseed rape isoforms 3, 4, 6, and 7belong to the second group. It is interesting that otherplant BCCPs from soybean and Mesembryanthemumcrystallinum sources formed a group distinct fromclass one and two Brassicaceae BCCPs. Relatednessof AtBCCP2 with class two oilseed rape BCCPsprompted the investigation of BCCPs from develop-ing seed. Oilseed rape developing seed contained atleast four BCCP polypeptides ranging in size from 22

to 37 kD. Analysis of developing oilseed rape seedfrom other laboratories also revealed multiple biotin-containing proteins in this size range, though thelower Mr forms were attributed to proteolysis (Elbor-ough et al., 1996). Since the sizes of these low molec-ular mass biotin-containing polypeptides (22 and25 kD) agree favorably with the apparent size ofAtBCCP2, we suggest they represent the class twoBCCPs rather than proteolytic derivatives of the 35-and 37-kD putative class one isoforms. This is sup-ported by direct evidence that oilseed rape contains atleast four genes that encode class two BCCP isoforms(Elborough et al., 1996) and like AtBCCP2, the oilseedrape genes appear to have evolved for expression inreproductive organs (Fig. 6B). The higher BCCP copynumber in oilseed rape compared with Arabidopsis islikely due to its allotetraploid genome. The potentialimportance of BCCPs in ACCase function and fattyacid biosynthesis is reflected not only in gene com-plexity, but by the observation that all BCCP isoformsfrom Arabidopsis and oilseed rape are up-regulatedmidway through seed development, coinciding withthe period of maximal oil accumulation (Turnham andNorthcote, 1983; Mansfield and Briarty, 1992).

In conclusion, two classes of BCCP genes exist inBrassicaceae plants. The data presented here demon-strate class one BCCPs are expressed in all tissuesexamined, whereas class two are expressed in repro-ductive organs, particularly developing seeds. Anunderstanding of class two BCCP isoforms and theirpossible specialized function during seed develop-ment awaits further biochemical and molecular ge-netic studies.

Figure 8. Dendrogram analysis of BCCPs. Clustal analysis was per-formed with Genetics Computer Group DNA analysis software (Mad-ison, WI) using default parameters. Length of horizontal lines indi-cates inverse degree of relatedness. Cyanobacterial and plantsequences are indicated. GenBank accession numbers not men-tioned in Figure 1 are X90727, X90728, X90729, X90731, andX90732, for oilseed rape isoforms 1, 2, 3, 6, and 7; AI822996,Mesembryanthemum; and U59235, Synechococcus.

Thelen et al.



MATERIALS AND METHODS

Plant Materials

All seeds were sown in a 1:1 mixture of water-saturatedvermiculite and peat moss-enriched soil. Arabidopsis (Co-lumbia ecotype) plants were grown at 18°C under low lightfluence (49 mmol photon m22 s21). Oilseed rape (Brassicanapus) was grown at 20°C during a 16-h photoperiod (294mmol photon m22 s21), and 15°C during the dark. Pea(Pisum sativum L. cv Little Marvel) seedlings were grown ata constant 22°C with an 8-h photoperiod (210 mmol photonm22 s21). For seed development analyses, flowers weretagged at the point when petals first appeared from theflower whorl.

Identification and Sequencing of AtBCCP2 cDNA

The AtBCCP1 cDNA (GenBank accession no. U23155)was used as a query of the Arabidopsis EST database(Newman et al., 1994) using the BLAST (BLAST, Altschul etal., 1990). A different class of BCCP cDNAs was observedas a result of this query. The cDNA that appeared to be thelongest at the 59 end (GenBank accession no. H37386) wasordered from the Arabidopsis Biological Research Center(Columbus, OH). Both strands of the cDNA were se-quenced by primer walking using 20-mer oligonucleotides.All other steps were performed according to the dye-deoxynucleotide chain termination procedure (AppliedBiosystems, Foster City, CA). Reaction products were an-alyzed on an ABI automated sequencer at the MichiganState University DNA core facility.

Northern-Blot Analyses

Total RNA was isolated from Arabidopsis organs usinga guanidinium thiocyanate extraction procedure (Sam-brook et al., 1989). RNA was quantitated by A260. RNAsamples were brought up to 15 mL with sterile water andequal volumes of formamide and formaldehyde wereadded prior to heating for 15 min at 50°C. Electrophoresisof RNA was performed in a 1% (w/v) agarose gel contain-ing 2.2 m formaldehyde and subsequently blotted to Hy-bond nylon membrane (Amersham, Buckinghamshire, UK)by capillary action for approximately 16 h. After transfer,membranes were rinsed briefly with 0.23 SSC, 0.1% (w/v)SDS, and were then dried at 80°C for 3 h. ImmobilizedrRNA was visualized by rehydrating the membrane in0.03% (w/v) methylene blue, 0.3 m sodium acetate, pH 6.0for 1 min. Excess stain was removed by iterative washeswith sterile water. Membranes were prehybridized for 6 hat 42°C in 15 mL of 50% (v/v) deionized formamide, 53SSPE, 0.5% (w/v) SDS, 53 Denhardt’s (503 5 1% [w/v] ofthe following: Ficoll, polyvinylpyrrolidone, bovine serumalbumin), 5% (w/v) dextran sulfate, and 100 mg mL21

sheared salmon sperm DNA. Hybridization was per-formed at 42°C for 16 h in the same solution using the 39one-half of BCCP1 (GenBank accession no. Z25714, 627–1,089 bp) or the 39-UTR of BCCP2 (BamHI, 871–1,235 bp)labeled by random hexamer extension. Subsequent to hy-

bridization, the membranes were washed once at 28°C with23 SSPE, 0.5% (w/v) SDS for 5 min then twice at 28°C for15 min. Final two washes were performed at 50°C with0.13 SSPE, 0.5% (w/v) SDS for 15 min each. Membraneswere covered with cellophane and exposed to autoradiog-raphy film. Hybridization signals were quantitated by Im-age Quant software (Molecular Dynamics, Sunnyvale, CA)accounting for area and signal intensity. Signals were ex-pressed as values relative to the highest signal on the blot.

Extraction of Arabidopsis Protein for SDS-PAGE andDetection of Biotin-Containing Polypeptides

Extraction of protein from roots, leaves, flowers, andsiliques was performed as follows. Up to 0.1 g of plantmaterial was harvested and placed into a 1.5-mL plasticmicrofuge tube. Plant material was pulverized with a mi-crocentrifuge pestle in the presence of liquid nitrogen.Powder was immediately reconstituted in 0.2 to 0.5 mL ofextraction buffer (0.2% [w/v] SDS, 8 m urea, and 2% [v/v]2-mercaptoethanol) by vortexing. Samples were thenheated at 95°C for 15 min. Insoluble debris was collected bycentrifugation for 15 min at 12,000g. Supernatant was re-moved and placed into a fresh microfuge tube. Seed pro-tein was extracted by placing the dissected seeds directlyinto extraction buffer and homogenizing with a plasticmicrofuge pestle. Insoluble debris was collected by centrif-ugation and the supernatant was saved for analysis. Pro-tein concentration was determined by dye-binding proteinassay using bovine serum albumin as the standard (Brad-ford, 1976). Equal amounts of total protein were resolvedby SDS-PAGE for immunoblot analyses.

SDS-PAGE and protein transfer to nitrocellulose wasperformed using standard conditions (Towbin et al., 1979).Nitrocellulose membranes were blocked for at least 1 hwith 10 mm Tris-HCl, pH 8.0, 0.15 m sodium chloride, 0.3%(v/v) Tween 20 (TBS-T), and 2% (w/v) nonfat dry milk.Anti-biotin antibodies conjugated to alkaline phosphatase(Kirkegaard and Perry Laboratories, Gaithersburg, MD)were directly added at a 1:2,000 dilution. The sensitivity ofthese antibodies (2 ng of purified recombinant BCCP) wasover 100-fold higher than similar products from variousmanufacturers. Probing proceeded for 16 h at 25°C afterwhich the membranes were briefly rinsed with TBS-T. Themembranes were then washed 6 times for 10 min each withTBS-T. Blots were then washed 5 min with developingsolution (0.1 m Tris-HCl, pH 9.5, 0.1 m sodium chloride,and 5 mm magnesium chloride) before colorimetric detec-tion in developing solution containing 0.33 mg mL21

p-nitro blue tetrazolium chloride and 0.17 mg mL21

5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt. Im-munoblot signals were quantitated using Image Quantsoftware, which accounted for band area plus intensity.Signals were corrected for background and expressed as avalue relative to the most abundant signal on the blot.

Chloroplast Import of AtBCCP2

Chloroplasts were isolated from 10-d-old pea leaves ac-cording to a previous procedure (Bruce et al., 1994). In vitro




transcription and translation was performed with the quick-coupled T7 polymerase rabbit reticulocyte lysate kit avail-able from Promega (Madison, WI). Lysate (5 mL) containingradiolabeled AtBCCP2 was added directly to 6 mL of 100 mmMgATP and 139 mL of import buffer (50 mm HEPES- [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] KOH, pH8.0, and 330 mm sorbitol). Isolated pea chloroplasts werediluted to 1 mg chlorophyll mL21 with import buffer and 50mL was added to diluted lysate mixture for a final volume of200 mL. Import proceeded for 30 min at 25°C after which thechloroplasts were diluted 5-fold with import buffer andcentrifuged at 1,500g for 3 min. The supernatant was dis-carded and the chloroplast pellet was gently washed in 1 mLof import buffer. The chloroplasts were collected by centrif-ugation and washed a second time. The washed chloroplastpellet was resuspended in 160 mL of import buffer and50-mL aliquots were placed into three microfuge tubes. Alltubes contained 1 mm calcium chloride, tube 2 and 3 con-tained 0.4 mg mL21 thermolysin protease (Sigma, St. Louis),and tube 3 also contained 0.05% (v/v) Triton X-100, all finalconcentrations. Protease digestion proceeded for 30 min at4°C and was terminated by the addition of 1 mL of importbuffer containing 5 mm EDTA and 5 mm EGTA. Intactchloroplasts were collected by centrifugation and washedwith 1 mL of import buffer, 5 mm EDTA, and 5 mm EGTA.The final chloroplast pellet was resuspended in 50 mL ofSDS-PAGE sample buffer (2% [w/v] SDS, 8 m urea, and 2%[v/v] 2-mercaptoethanol) and heated at 95°C for 15 minprior to loading onto SDS-PAGE.

Recombinant Expression of Arabidopsis BCCPs and BC

Oligonucleotide primers were designed to PCR amplifythe mature ORF of AtBCCP1 and AtBCCP2 for subcloninginto pET28b expression vector (Novagen, Madison, WI).Based upon the experimentally determined processing sitefor soybean BCCP, primers were designed to start withSer-64 (AtBCCP1) or Thr56 (AtBCCP2). Primers JO625, gcgt-gaGAATTCcatgggctctaacaaggttagtactggtgca (59-sense), andJO634, gcgaagCTCGAGtacggttgaaccacaaacagagg (39-anti-sense), were used to amplify AtBCCP1 mature ORF (nucle-otides 245–898). Primers JO627, ccctaTCTAGAGAATTCag-aaggagatatgggcactaatgaggttgtttctaac (59-sense), and JO628,gcgaagCTCGAGtcaaggtgcgatgacaaaaag (39-antisense), wereused to PCR amplify the AtBCCP2 mature ORF (nucleotides258–861). Primer JO627 contained an artificial ribosomalbinding site upstream of an ATG start codon (noted initalics) to initiate translation for the mature AtBCCP2polypeptide without a poly (His) tag. Primers JO685, gcgt-gaGAATTCtgcagtggtggtgataagatt (59-sense), and JO686,gggtcaCTCGAGctaaaccgttgcgtttgtcagatc (39-antisense), wereused to amplify the AtBC ORF (nucleotides 329–1732). PCRwas performed under standard conditions with plasmidDNA as template (Thelen et al., 1999). The AtBCCP1 ampli-con was subcloned into the EcoRI and XhoI sites of pET28bto obtain an amino-terminal poly-(His) fusion-tagged fusionprotein. AtBCCP2 amplicon was subcloned into the XbaI,XhoI ,and EcoRI, XhoI sites of pET28b to obtain matureprotein without and with an amino-terminal poly-(His) fu-

sion tag, respectively. AtBC amplicon was subcloned intothe EcoRI and XhoI sites of pET28a to obtain an amino-terminal poly (His) fusion.

To amplify the mature ORF of AtBCCP1, a 39 primer wasinitially designed according to the defined ORF of Gen-Bank sequence U23155 (CAC1, Choi et al., 1995). Whenused in combination with the 59 primer, no amplicon wasobtained by RT-PCR or PCR using total RNA and plasmidDNA as template, respectively. As a consequence, the nu-cleotide sequence of U23155 was verified against two ESTsequences (AI999240 and Z25714) and chromosome Vgenomic sequence (AB005242). A frameshift at nucleotide895, due to an extra cytosine at this position, was revealed.This resulted in a GTA read-through, instead of a TAG stopcodon, and a subsequent -VESAP carboxy-terminal tail inthe deduced amino acid sequence (Fig. 1). The gene se-quence for AtBCCP1 also contains a frameshift mutation atthis same position (U62029, Ke et al., 1997). To check forany additional differences, the full-length AtBCCP1 EST(AI999240) was completely sequenced. Besides the frame-shift mutation, clone AI999240 contained 37 and 124 bplonger 59- and 39-UTRs, respectively. Due to the significantdifferences between these two AtBCCP1 cDNAs, the fullsequence for clone AI999240 was submitted to GenBank(assigned accession no. AF236873). To proceed with PCRamplification of AtBCCP1, the antisense primer was rede-signed to account for the aforementioned frameshift.

Poly (His)-tagged recombinant protein was expressed andpurified from the protease-deficient BL21(DE3) E. coli cell lineaccording to standard conditions (Thelen et al., 1998), exceptfor the inclusion of 0.2 mm biotin throughout the growth andinduction phases for BCCPs. Protein was dialyzed in 2 L of 10mM TES-KOH, pH 7.4, 10% (v/v) glycerol, and 1 mm dithio-threitol for 16 h at 4°C and was subsequently concentrated onultrafiltration membranes with a 5-kD cutoff.

Reconstitution of BC Half-Reaction with RecombinantBC and BCCPs

Biotin carboxylase activity was assayed as the enzymatictransfer of 14CO2 from NaH14CO3 to free d-biotin (Sigma)or purified, recombinant BCCPs to form carboxybiotin,which is stable to unlabeled CO2 bubbling. Reaction con-ditions were similar to those described previously (Guchaitet al., 1974) except for the use of 100 mm HEPES-KOH, pH8.0, 4 mm MgCl2, and 3 mm dithiothreitol in reaction me-dium. Reactions (150 mL) were initiated with rBC enzyme,proceeded for 30 min at 25°C, and were terminated bypipetting the reaction into 2 mL of ice-cold water contain-ing 200 mL of 1-octanol. Unlabeled CO2 was then bubbledinto the reaction vial for 30 min at 4°C. After gasing, 0.2 mLof 0.1 n NaOH was added prior to liquid scintillationspectroscopy. Minus enzyme background rates were deter-mined for each experiment and were typically less than 5%of the actual rate. The specific activity of NaH14CO3 inreaction was approximately 3,000 dpm nmol21 NaH14CO3

and was verified for each experiment. All values are themean of at least three reactions. Use of BCCPs as carboxyacceptors was assayed in triplicate using independent pro-tein preparations.

Thelen et al.



DEAE Chromatography of Arabidopsis Silique Extract

Arabidopsis siliques were homogenized in a mortar andpestle with liquid nitrogen and reconstituted in homogeniza-tion buffer, 50 mm HEPES-KOH, pH 8.0, 10% (v/v) glycerol,1% (v/v) Triton X-100, 2 mm phenylmethanesulfonyl fluo-ride, 2 mm benzamidine, 2 mm e-amino-n-caproic acid, 5 mmEDTA, and 14 mm 2-mercaptoethanol by vortexing for 30 s.Particulate matter was removed by centrifugation, 14 K g for1 min and supernatant was applied to a DEAE-sepharosematrix (0.6-mL bed volume) pre-equilibrated with homoge-nization buffer. Column was washed with three volumes ofhomogenization buffer (minus glycerol and Triton X-100) andeluted with a sodium chloride gradient in wash medium.Elution fractions were heated in sample buffer and resolvedby SDS-PAGE for immunoblotting with anti-castor BC(Roesler et al., 1997) and anti-biotin antibodies.

ACKNOWLEDGMENT

The authors thank the Arabidopsis Biological ResourceCenter for providing the cDNA clones used in thisinvestigation.

Received September 7, 2000; returned for revision Novem-ber 1, 2000; accepted November 17, 2000.

LITERATURE CITED

Alban C, Baldet P, Douce R (1994) Localization and char-acterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one issensitive to aryloxyphenoxyproprionate herbicides. Bio-chem J 300: 557–565

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ(1990) Basic local alignment search tool. J Mol Biol 215:403–410

Bao X, Shorrosh BS, Ohlrogge J (1997) Isolation and char-acterization of an Arabidopsis biotin carboxylase geneand its promoter. Plant Mol Biol 35: 539–550

Blanchard CZ, Chapman-Smith A, Wallace JC, WaldropGL (1999) The biotin domain peptide from the biotincarboxyl carrier protein of Escherichia coli acetyl-CoAcarboxylase causes a marked increase in the catalyticefficiency of biotin carboxylase and carboxyltransferaserelative to free biotin. J Biol Chem 274: 31767–31769

Bradford MM (1976) A rapid and sensitive method for thequantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254

Brocklehurst SM, Perham RN (1993) Prediction of thethree-dimensional structure of the biotinylated domainfrom yeast pyruvate carboxylase and of the lipoylatedH-protein from the pea leaf glycine cleavage system: anew automated method for the prediction of proteintertiary structure. Protein Sci 2: 626–639

Bruce BD, Perry S, Froehlich J, Keegstra K (1994) In vitroimport of proteins into chloroplasts. Plant Mol Biol ManJ1: 1–15

Choi JK, Yu F, Wurtele E, Nikolau BJ (1995) Molecularcloning and characterization of the cDNA coding for thebiotin-containing subunit of the chloroplastic acetyl-coenzyme A carboxylase. Plant Physiol 109: 619–625

Duval M, Job C, Alban C, Douce R, Job D (1994) Devel-opmental patterns of free and protein-bound biotinduring maturation and germination of seeds of Pisumsativum: characterization of a novel seed-specific biotin-ylated protein. Biochem J 299: 141–150

Elborough KM, Winz R, Deka RK, Markham JE, WhiteAJ, Rawsthorne S, Slabas AR (1996) Biotin carboxylcarrier protein and carboxyltransferase subunits of themulti-subunit form of acetyl-CoA carboxylase from Bras-sica napus: cloning and analysis of expression duringoilseed rape embryogenesis. Biochem J 315: 103–112

Evans DE, Taylor PE, Singh MB, Knox RB (1992) Theinterrelationship between the accumulation of lipids, pro-tein and the level of acyl carrier protein during the devel-opment of Brassica napus L. pollen. Planta 186: 343–354

Fatland BL, Qian H, Nikolau BJ, Wurtele E (1999) Anti-sense expression of the CAC1 subunit of ACCase in Ara-bidopsis results in significant modifications in leaf and cellorganization (abstract no. 276). Plant Physiol Plant BiolSuppl 78

Girke T, Todd J, Ruuska S, White J, Benning C, OhlroggeJ (2000) Microarray analysis of developing Arabidopsisseeds. Plant Physiol 124: 1570–1581

Guchait RB, Polakis SE, Dimroth P, Stoll E, Moss J, LaneMD (1974) Acetyl coenzyme A carboxylase system ofEscherichia coli: purification and properties of the biotincarboxylase, carboxyltransferase, and carboxyl carrierprotein components. J Biol Chem 249: 6633–6645

Hlousek-Radojcic A, Post-Beittenmiller D, Ohlrogge JB(1992) Expression of constitutive and tissue-specific acylcarrier protein isoforms in Arabidopsis. Plant Physiol 98:206–214

Hunter SC, Ohlrogge JB (1998) Regulation of spinach chlo-roplast acetyl-CoA carboxylase. Arch Biochem Biophys359: 170–178

Kannangara CG, Stumpf PK (1972) Fat metabolism inhigher plants: a prokaryotic type acetyl-CoA carboxylasein spinach chloroplasts. Arch Biochem Biophys 152: 83–91

Ke J, Choi JK, Smith M, Horner HT, Nikolau BJ, WurteleES (1997) Structure of the CAC1 gene and in situ char-acterization of its expression: the Arabidopsis thalianagene coding for the biotin-containing subunit of the plas-tidic acetyl-coenzyme A carboxylase. Plant Physiol 113:357–365

Li S-J, Cronan JE Jr (1992a) The gene encoding the biotincarboxylase subunit of Escherichia coli acetyl-CoA carbox-ylase. J Biol Chem 267: 855–863

Li S-J, Cronan JE Jr (1992b) The genes encoding the twocarboxylase subunits of Escherichia coli acetyl-CoA car-boxylase. J Biol Chem 267: 16841–16847

Li S-J, Cronan JE Jr (1992c) Putative zinc finger proteinencoded by a conserved chloroplast gene is very likely asubunit of a biotin-dependent carboxylase. Plant MolBiol 20: 759–761

Mansfield SG, Briarty LG (1992) Cotyledon cell develop-ment in Arabidopsis thaliana during reserve deposition.Can J Bot 70: 151–164

Marini P, Li SJ, Gardiol D, Cronan JE, de Mendoza D(1995) The genes encoding the biotin carboxyl carrierprotein and biotin carboxylase subunits of Bacillus subti-




lis acetyl coenzyme A carboxylase, the first enzyme offatty acid synthesis. J Bacteriol 177: 7003–7006

Mekhedov S, Martinez de Ilarduya O, Ohlrogge JB (2000)Toward a functional catalog of the plant genome: a surveyof genes for lipid biosynthesis. Plant Physiol 122: 389–401

Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H,McIntosh L, Ohlrogge J, Raikhel N, Somerville S, Tho-mashow M et al. (1994) Genes galore: a summary ofmethods for accessing results from large-scale partialsequencing of anonymous Arabidopsis cDNA clones.Plant Physiol 106: 1241–1255

Nikolau BJ, Hawke C (1984) Purification and characteriza-tion of maize leaf acetyl-coenzyme A carboxylase. ArchBiochem Biophys 228: 85–96

Ohlrogge JB, Jaworski JG (1997) Regulation of fatty acidbiosynthesis. Annu Rev Plant Physiol Plant Mol Biol 48:109–136

Ohlrogge JB, Kuhn DN, Stumpf PK (1979) Subcellularlocalization of acyl carrier protein in leaf protoplasts ofSpinacia oleracea. Proc Natl Acad Sci USA 78: 1194–1198

Page RA, Okada S, Harwood JL (1994) Acetyl-CoA car-boxylase exerts strong flux control over lipid synthesis inplants. Biochim Biophys Acta 1210: 369–372

Post-Beittenmiller MA, Jaworski JG, Ohlrogge JB (1991)In vivo pools of free and acylated acyl carrier proteins inspinach: evidence for sites of regulation of fatty acidbiosynthesis. J Biol Chem 266: 1858–1865

Post-Beittenmiller MA, Roughan G, Ohlrogge JB (1992)Regulation of plant fatty acid biosynthesis: analysis ofacyl-CoA and acyl-acyl carrier protein substrate pools inspinach and pea chloroplast. Plant Physiol 100: 923–930

Reverdatto S, Beilinson V, Nielsen NC (1999) A multisub-unit acetyl-coenzyme A carboxylase from soybean. PlantPhysiol 119: 961–978

Roesler KR, Savage LJ, Shintani DK, Shorrosh BS, Ohl-rogge JB (1996) Co-purification, co-immunoprecipitation,and coordinate expression of acetyl-coenzyme A carbox-ylase activity, biotin carboxylase, and biotin carboxyl car-rier protein of higher plants. Planta 198: 517–525

Roesler KR, Shintani D, Savage L, Boddupalli S, Ohl-rogge JB (1997) Targeting of the Arabidopsis homomericacetyl-coenzyme A carboxylase to Brassica napus seedplastids. Plant Physiol 113: 75–81

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Clon-ing: A Laboratory Manual. Cold Spring Harbor Labora-tory Press, Cold Spring Harbor, NY

Samols D, Thornton CG, Murtif VL, Kumar GK, HaaseFC, Wood HG (1988) Evolutionary conservation amongbiotin enzymes. J Biol Chem 263: 6461–6464

Sasaki Y, Hakamada K, Suama Y, Nagano Y, Furusawa I,Matsuno R (1993) Chloroplast-encoded protein as a sub-unit of acetyl-CoA carboxylase in pea plant. J Biol Chem268: 25118–25123

Sasaki Y, Kozaki A, Hatano M (1997) Link between lightand fatty acid synthesis: thioredoxin-linked reductiveactivation of plastidic acetyl-CoA carboxylase. Proc NatlAcad Sci USA 94: 11096–11101

Sasaki Y, Konishi T, Nagano Y (1995) The compartmenta-tion of acetyl-coenzyme A carboxylase in plants. PlantPhysiol 108: 445–449

Savage LJ, Ohlrogge JB (1999) Phosphorylation of peachloroplast acetyl-CoA carboxylase. Plant J 18: 521–527

Schulte W, Topfer R, Stracke R, Schell J, Martini N (1997)Multi-functional acetyl-CoA carboxylase from Brassicanapus is encoded by a multi-gene family: indication forplastidic localization of at least one isoform. Proc NatlAcad Sci USA 94: 3465–3470

Schutt BS, Brummel M, Schuch R, Spener F (1998) Therole of acyl carrier protein isoforms from Cuphea lanceo-lata seeds in the de novo biosynthesis of medium-chainfatty acids. Planta 205: 263–268

Shintani DK, Ohlrogge JB (1994) The characterization of amitochondrial acyl carrier protein isoform isolated fromArabidopsis thaliana. Plant Physiol 104: 1221–1229

Shintani DK, Ohlrogge JB (1995) Feedback inhibition offatty acid synthesis in tobacco suspension cells. Plant J 7:577–587

Shorrosh BS, Dixon RA, Ohlrogge JB (1994) Molecular clon-ing, characterization, and elicitation of acetyl-CoA carbox-ylase from alfalfa. Proc Natl Acad Sci USA 91: 4323–4327

Shorrosh BS, Savage LJ, Soll J, Ohlrogge JB (1996) Thepea chloroplast membrane-associated protein, IEP96, is asubunit of acetyl-CoA carboxylase. Plant J 10: 261–268

Song J, Wurtele ES, Nikolau BJ (1994) Molecular cloningand characterization of the cDNA coding for the bio-tin-containing subunit of 3-methylcrotonoyl-CoA car-boxylase: identification of the biotin carboxylase andbiotin-carrier domains. Proc Natl Acad Sci USA 91:5779–5783

Suh MC, Schultz DJ, Ohlrogge JB (1999) Isoforms of acylcarrier protein involved in seed-specific fatty acid syn-thesis. Plant J 17: 679–688

Sun J, Ke J, Johnson JL, Nikolau BJ, Wurtele ES (1997)Biochemical and molecular biological characterization ofCAC2, the Arabidopsis thaliana gene coding for the biotincarboxylase subunit of the plastidic acetyl-coenzyme Acarboxylase. Plant Physiol 115: 1371–1383

Thelen JJ, Muszynski MG, David NR, Luethy MH, El-thon TE, Miernyk JA, Randall DD (1999) The dihydro-lipoamide S-acetyltransferase subunit of the mitochon-drial pyruvate dehydrogenase complex from maizecontains a single lipoyl domain. J Biol Chem 274:21769–21775

Thelen JJ, Muszynski MG, Miernyk JA, Randall DD(1998) Molecular analysis of two pyruvate dehydroge-nase kinases from maize. J Biol Chem 273: 26618–26623

Towbin H, Staehelin T, Gordon J (1979) Electrophoretictransfer of proteins from polyacrylamide gels to nitrocel-lulose sheets: procedure and some applications. ProcNatl Acad Sci USA 76: 4350–4354

Turnham E, Northcote DH (1983) Changes in the activityof acetyl-CoA carboxylase during rape-seed formation.Biochem J 212: 223–229

White JA, Todd J, Newman T, Focks N, Girke T, Martinezde Ilarduya O, Jaworski JG, Ohlrogge J, Benning C(2000) A new set of Arabidopsis ESTs from developingseeds: the metabolic pathway from carbohydrates to seedoil. Plant Physiol 124: 1582–1594

Thelen et al.



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