Identification of Mitochondrial Coenzyme A Transportersfrom Maize and Arabidopsis1[W][OA]

Rémi Zallot2, Gennaro Agrimi2, Claudia Lerma-Ortiz, Howard J. Teresinski, Océane Frelin,Kenneth W. Ellens, Alessandra Castegna, Annamaria Russo, Valérie de Crécy-Lagard, Robert T. Mullen,Ferdinando Palmieri, and Andrew D. Hanson*

Microbiology and Cell Science Department (R.Z., C.L.-O., V.d.C.-L.) and Horticultural Sciences Department(O.F., K.W.E., A.D.H.), University of Florida, Gainesville, Florida 32611; Laboratory of Biochemistry andMolecular Biology, Department of Biosciences, Biotechnologies, and Biopharmaceutics, University of Bari,70125 Bari, Italy (G.A., A.C., A.R., F.P.); and Department of Molecular and Cellular Biology, University ofGuelph, Guelph, Ontario, Canada N1G 2W1 (H.J.T., R.T.M.)

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes,implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plantmitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrierfamily, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparativegenomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereasnonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) andArabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Dmitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteinshave CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, andsubcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) BrightYellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitousimportance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughoutthe plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially locatedmitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliableannotation in other angiosperm genomes.

CoA acts as an acyl carrier in many reactions ofprimary and secondary metabolism, and some 8% ofthe nearly 4,900 enzymes described in the EnzymeCommission database are CoA dependent (Bairoch, 2000).CoA occupies a central position in lipid metabolism,

respiration, gluconeogenesis, and other pathways (Leonardiet al., 2005). It is present in all forms of life, but while allorganisms can synthesize it from pantothenate (vitamin B5),only prokaryotes, plants, and fungi are able to synthesizepantothenate; animals obtain pantothenate from the diet(Daugherty et al., 2002; Leonardi et al., 2005; Webb andSmith, 2011).

In plants, the steps that convert pantothenate to CoAare almost certainly cytosolic (Webb and Smith, 2011;Gerdes et al., 2012). CoA, however, is required in mi-tochondria for the citric acid cycle, in chloroplasts forfatty acid synthesis, and in peroxisomes for b-oxidation.CoA, therefore, must be imported into these organellesfrom the cytosol, and indeed, early work demonstrateda CoA transport system in potato (Solanum tuberosum)mitochondria (Neuburger et al., 1984). Yeast (Saccharomycescerevisiae) and mammalian mitochondria and peroxi-somes likewise import CoA because they cannot makeit (Fiermonte et al., 2009; Agrimi et al., 2012b). Thecompartmentation of CoA in all eukaryotes appearsto be closely regulated, with cytosol and organellesmaintaining separate CoA pools whose levels canmodulate fluxes through CoA-dependent reactions(Hunt and Alexson, 2002; Leonardi et al., 2005; DeMarcos Lousa et al., 2013).

1 This work was supported by the National Science FoundationPlant Genome Research Program (grant no. IOS–1025398 to A.D.H.),by the Ministero dell’Università e della Ricerca, the Center of Excel-lence in Genomics, and the Italian Human ProteomeNet (grant no.RBRN07BMCT_009 to F.P.), by the National Sciences and Engineer-ing Research Council of Canada (grant no. 217291 to R.T.M.), and byan endowment from the C.V. Griffin Sr. Foundation. R.T.M. holds aUniversity of Guelph Research Chair, and H.J.T. is supported by aUniversity of Guelph College of Biological Science Faculty ResearchAssistance Award.

2 These authors contributed equally to the article.* Corresponding author; e-mail adha@ufl.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Andrew D. Hanson (adha@ufl.edu).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

tion.www.plantphysiol.org/cgi/doi/10.1104/pp.113.218081

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Figure 1. Phylogenetic analysis of known andpredicted CoA transporters. A, Phylogenetictree of the experimentally validated CoA trans-porters from yeast, human, and Arabidopsis,the candidate CoA transporters from Arabi-dopsis (At1g14560 and At4g26180) and maize(GRMZM2G420119 and GRMZM2G161299;abbreviated to ZM420119 and ZM161299), andall other Arabidopsis MCF proteins. Sequenceswere aligned with ClustalW; the tree was con-structed by the neighbor-joining method withMEGA5. Bootstrap values (%) for 1,000 replicatesare shown next to nodes; those of less than 50%are omitted. Only the tree topology is shown. Theyellow highlighted sector contains the validatedhuman and yeast mitochondrial CoA transportersand their Arabidopsis and maize homologs; thetype 1 and type 2 plant carriers are indicated. Thegreen highlighted sector contains the validatedhuman and Arabidopsis peroxisomal CoA trans-porters. B, Phylogenetic distribution of type 1 andtype 2 predicted CoA transporters among angio-sperms, gymnosperms, and lower plants. Redboxes and blue boxes indicate the number of type1 and type 2 sequences, respectively, representedin each genome or transcriptome. Piebald red/blue boxes indicate homologs in lower plants thatcould not be assigned to either type 1 or type 2.

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Mitochondrial CoA transporters belonging to themitochondrial carrier family (MCF) have been identi-fied in yeast (Leu-5p; Prohl et al., 2001) and human(SLC25A42; Fiermonte et al., 2009). Furthermore,peroxisomal CoA carriers from human (SLC25A17;Agrimi et al., 2012b) and Arabidopsis (Arabidopsisthaliana; peroxisomal CoA and NAD carrier [PXN];Agrimi et al., 2012a) have also been identified. How-ever, no transporters for CoA are known for plantmitochondria or chloroplasts (Palmieri et al., 2011;Gerdes et al., 2012).In this study, a comparative genomic analysis first

identified close Arabidopsis and maize (Zea mays)homologs of the yeast and mammalian mitochon-drial CoA carriers as candidates for the missing plantmitochondrial or chloroplast transporters. Experi-mental evidence then demonstrated that the candi-date proteins transport CoA when expressed in yeast,that they are targeted to mitochondria in vitro and inplanta, and that they are expressed throughout theplant.

RESULTS

Identification of Candidate Plant CoA Carriers

An earlier analysis of Arabidopsis, human, and yeastMCF proteins identified At1g14560 and At4g26180 aspotential CoA carriers, based on the presence of se-quence motifs (symmetry-related amino acid triplets)similar to those in known, mitochondrially localizedCoA carriers (Palmieri et al., 2011). At1g14560 andAt4g26180 share only 59% amino acid identity and soare substantially diverged.BLAST searches of the maize genome using each Arab-

idopsis sequence as query detected GRMZM2G420119and GRMZM2G161299 (henceforth ZM420119 andZM161299) as the closest homologs (66%–67% iden-tity) of At1g14560 and At4g26180, respectively. Allfour proteins have the tripartite structure that charac-terizes the MCF (Palmieri et al., 2011) and are predictedto contain the usual six transmembrane domains

(Supplemental Fig. S1). Phylogenetic analyses of theseproteins, the human and yeast mitochondrial CoAcarriers, and all Arabidopsis, yeast, and human MCFproteins consistently placed all four candidates and theyeast and human CoA carriers in the same clade. Thus,among all 58 Arabidopsis MCF proteins, those closest

Figure 2. Complementation of the growth phe-notype of the yeast CoA carrier deletant leu5Δ bythe expression of candidate plant CoA carriers.Serial dilutions of wild-type (WT) cells harboringpYES2/CT (vector) or leu5Δ cells harboring pYES2/CT alone or encoding maize (ZM420119 andZM161299) or Arabidopsis (At4g26180 andAt1g14560) proteins were plated on YP mediumcontaining 3% glycerol and incubated for 72 hat 30˚C. pYES2/CT constructs encoding the humanCoA transporter SLC25A42 or the ArabidopsisNAD+ transporter AtNDT1 were included as positiveand negative controls, respectively.

Figure 3. Effects of the expression of candidate plant CoA carriers onmitochondrial CoA contents of leu5D yeast cells. Cells were culturedin liquid YP medium containing 2% ethanol. Mitochondria were iso-lated from wild-type (WT) cells harboring pYES2/CT (vector) or fromleu5D cells harboring pYES2/CT alone or encoding At1g14560,At4g26180, ZM420119, or ZM161299. pYES2/CT constructs encodingthe human CoA transporter SLC25A42 or the human pyrimidine nu-cleotide transporter SLC25A33 were included as positive and negativecontrols, respectively. Data are means and SE of at least three inde-pendent experiments and were subjected to one-way ANOVA fol-lowed by Bonferroni post hoc tests. Differences in CoA contentbetween leu5D cells harboring pYES2/CT and other strains that aresignificant are indicated with asterisks: *P , 0.05, **P , 0.01, ***P ,0.001; ns, not significant.

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to the known mitochondrially localized CoA carriersare the candidates (Fig. 1A), and among all yeast orhuman MCF proteins, the candidates are closest tothe known mitochondrial CoA carriers (SupplementalFig. S2). The recently identified peroxisomal CoAcarriers from human (Agrimi et al., 2012b) and Arab-idopsis (Agrimi et al., 2012a) occupy a very differentclade (Fig. 1A).

The phylogenetic trees all separate the four can-didate genes into two types: type 1 composed ofAt1g14560 and ZM420119, and type 2 composed ofAt4g26180 and ZM161299 (Fig. 1A; Supplemental Fig.S2). One or two genes of each type were found in allother eudicotyledon and monocotyledon genomes andtranscriptomes examined as well as in that of the basalangiosperm Amborella trichopoda (Fig. 1B; SupplementalFig. S3). In contrast, gymnosperms had type 1 but nottype 2 sequences, whereas lower plants each had asingle sequence that could not be confidently assignedto either type (Fig. 1B; Supplemental Fig. S3).

Functional Complementation of the Yeast leu5D Mutant

Deletion of the yeast LEU5 gene, encoding a mito-chondrial CoA transporter, results in strongly retardedgrowth on rich medium containing a nonfermentablecarbon source, and expression of a functional CoAcarrier protein in the deletant (leu5D) strain restoresnormal growth (Prohl et al., 2001). The human Leu-5phomolog, SLC25A42, used as a positive control, re-stored normal growth on 1% yeast extract, 2% bacto-peptone (YP) medium plates containing 3% glycerol,as expected (Fiermonte et al., 2009). Similarly, allfour candidate plant CoA transporters, At1g14560,At4g26180, ZM420119, and ZM161299, restored nor-mal growth, as opposed to little or no growth for cellstransformed with vector alone or a negative control(the Arabidopsis NAD+ carrier AtNDT1; Fig. 2). Similarresults were obtained in liquid medium (SupplementalFig. S4). These data indicate that the plant type 1 andtype 2 proteins have CoA transport activity and thatthey are targeted to mitochondria in yeast. Furthermore,because the yeast Leu-5p protein is known to be in themitochondrial inner membrane (Prohl et al., 2001), theobserved complementation by the plant proteins indi-cates that they are likewise, at least in part, localized inthe inner membrane when expressed in yeast.

Complementation Restores Mitochondrial CoA Levels inthe leu5D Strain

The CoA content in mitochondria of the leu5D strainis severely reduced compared with the wild type,reflecting the role of Leu-5p in mitochondrial CoAimport (Prohl et al., 2001). The candidate proteins frommaize and Arabidopsis complemented this biochemi-cal phenotype of the leu5D strain: expression of thetype 1 candidates At1g14560 and ZM420119 fully

restored mitochondrial CoA levels to that of the wildtype; expression of the type 2 candidates At4g26180and ZM161299 restored levels to over one-half that ofthe wild type (Fig. 3). These data reinforce the con-clusion that the plant proteins mediate CoA transportinto yeast mitochondria.

Subcellular Localization

Organellar targeting of the four CoA carrier candi-dates was investigated using dual-import assays, inwhich labeled proteins are incubated with a mixture ofpurified pea (Pisum sativum) mitochondria and chlo-roplasts and then reisolated (Rudhe et al., 2002). Soy-bean (Glycine max) alternative oxidase (Rudhe et al.,2002) and Arabidopsis YgfZ protein (Waller et al.,2012) were used as positive controls for mitochondrialand chloroplast import, respectively. All four candi-dates behaved similarly to the mitochondria-targetedcontrol protein (i.e. the reisolated mitochondria treatedwith thermolysin contained a labeled band corre-sponding to the imported protein; Fig. 4), which in-dicates an intramitochondrial location and is consistentwith the complementation evidence above for an innermembrane location. The observed lack of difference

Figure 4. Protein uptake by purified pea mitochondria and chloro-plasts in dual-import assays. The four CoA carrier cDNAs, plus theArabidopsis YgfZ (AtYgfZ) and soybean alternative oxidase (GmAox)cDNAs as positive controls for chloroplast and mitochondrial import,respectively, were transcribed and translated in vitro in the presence of[3H]Leu. The translation products (TP) were incubated in the light withmixed pea chloroplasts (C) and mitochondria (M). The organelles weremock treated (2) or thermolysin treated (+) to remove adsorbed pro-teins and then reisolated on a Percoll gradient. Proteins were separatedby SDS-PAGE and visualized by fluorography. Samples were loaded onthe basis of equal chlorophyll or mitochondrial protein content next toan aliquot of the translation product. Exposure times were adjusted togive comparable band intensities in all tracks. Molecular masses areindicated on the right.

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between the molecular mass of the precursors and theimported proteins is expected, because MCF proteinstypically lack cleavable targeting peptides (Palmisanoet al., 1998; Zara et al., 2009). None of the candidatesmirrored the behavior of the chloroplast-targeted controlprotein (Fig. 4).As a second approach for investigating subcellular

localization, tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, serving as a model plant cell system forstudying protein localization (Brandizzi et al., 2003;Miao and Jiang, 2007), were transiently transformedwith each of the four candidates linked at their N or Ctermini to GFP and examined by epifluorescence mi-croscopy. The expressed C-terminal fusions of bothmaize proteins and both Arabidopsis proteins showeda fluorescence pattern that colocalized with thatattributable to the endogenous mitochondrial inner

membrane marker, the b-ATPase subunit (Fig. 5).Notably, the fusion proteins often yielded a torus-likefluorescence pattern that circled the more punctatefluorescence attributable to the b-ATPase (Fig. 5,highly magnified images), suggesting that they maylocalize to two distinct subdomains of the inner mito-chondrial membrane, the inner boundary membraneand the cristae membrane (for review, see Logan, 2006;Mannella, 2006). Thus, the toroid fluorescence patternexhibited by the candidate fusion proteins in BY-2 cellsresembles that of several yeast GFP-tagged proteinsthat preferentially localize to the inner boundarymembrane, which lies parallel to the mitochondrialouter membrane (Wurm and Jakobs, 2006; Suppanzet al., 2009). The punctate fluorescence pattern ex-hibited by endogenous b-ATPase in BY-2 cells, on theother hand, is consistent with the localization of its

Figure 5. Representative epifluorescence images of tobacco BY-2 cells transiently expressing maize or Arabidopsis CoA carriercandidates C-terminally fused to GFP. Cells were biolistically bombarded with plasmid DNA encoding a GFP-tagged candidate.Approximately 4 h later, cells were formaldehyde fixed, permeabilized with pectolyase and Triton X-100, immunostained forendogenous mitochondrial b-ATPase, serving as a mitochondrial marker protein, and then examined by epifluorescence mi-croscopy. As indicated by the labels, each row of images corresponds to the fluorescence attributable to the candidate fusionprotein and endogenous b-ATPase immunostaining (green and red, respectively) and the corresponding merged image. Boxesrepresent the portion of the cell shown at higher magnification in the panels to the right. Arrowheads indicate examples oftoroidal structures containing a candidate fusion protein enclosing a punctate structure containing b-ATPase. Bar = 10 mm.

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yeast counterpart in the cristae membrane, which ex-tends into the matrix (Wurm and Jakobs, 2006). Thereare, however, other possible explanations for the con-centration of GFP-tagged inner membrane proteins atthe mitochondrial rim (Donzeau et al., 2000), so thisquestion needs to be studied in more detail.

Unlike the C-terminal fusions, the N-terminal GFPfusions for all four proteins localized only partially tomitochondria and mostly to the cytosol (data notshown), suggesting that the N-terminally appendedGFP moiety interfered with the mitochondrial target-ing information in the CoA carriers. Similar interfer-ence with mitochondrial targeting was observed forN-terminal fusions of GFP to thiamin diphosphatecarriers (Frelin et al., 2012). Finally, there was no ap-parent targeting of either the C- or N-terminal GFPfusion protein to plastids or peroxisomes. Together,the results of dual-import assays and of cell imagingdemonstrate the targeting of all four candidate pro-teins to plant mitochondria and not other organelles.

Expression Patterns of CoA Transporter Genes

The relative levels of the ZM161299 and ZM420119mRNAs were determined by quantitative reversetranscription-PCR in 10 tissues representing the wholeadult maize plant (Fig. 6). Both genes are expressed inall tissues tested, with fairly little variation (maximumof 4.2-fold). Their expression patterns are similar, ex-cept that the type 1 gene ZM420119 predominates insilks and the type 2 gene ZM161299 predominates intassels. Analysis of expression data for both maizegenes from genome-wide RNA-seq data sets gave asimilar picture (Supplemental Fig. S5A). Publicly avail-able microarray data for the Arabidopsis genes like-wise show expression throughout the plant, withrelatively little variation except in stamens and pollen,and with the type 1 gene At1g14560 always predomi-nant (Supplemental Fig. S5B). The ubiquitous expres-sion of both types of CoA carrier gene in both speciesis consistent with the universal requirement of mito-chondria for CoA, which is essential, among otherthings, for the oxidation of pyruvate via the tricar-boxylic acid cycle.

DISCUSSION

The bioinformatic prediction that the Arabidopsis andmaize homologs of the yeast and human mitochondrialCoA carriers themselves transport CoA is validated bythe evidence that they functionally complement thegrowth defect of the yeast leu5D mutant and restore itsmitochondrial CoA level. Therefore, we propose thatthese proteins be designated AtCoAc1 (At1g14560),AtCoAc2 (At4g26180), ZmCoAc1 (GRMZM2G420119),and ZmCoAc2 (GRMZM2G161299).

That these carriers belong to the MCF (Palmieri,2013) does not necessarily mean that they are targeted

to mitochondria, as several cases of plastid (Bedhommeet al., 2005; Kirchberger et al., 2008) or peroxisomal(Agrimi et al., 2012a, 2012b; Bernhardt et al., 2012)targeting are known. Nor does the fact that the plantproteins are targeted to mitochondria in yeast (i.e.they replace the missing mitochondrial CoA carrierin the leu5D mutant) prove that they are also targetedto mitochondria in planta. However, the results of invitro dual-import assays and of in vivo localizationof GFP fusion proteins provide strong evidence formitochondrial (inner membrane) localization for bothtype 1 and type 2 transporters. As neither type showedany plastid targeting in vitro or in vivo, they areprobably not responsible for CoA import into plas-tids. Thus, plastidial CoA transporters still remain tobe identified.

That homologs of both types of mitochondrial CoAtransporter occur in all the sequenced angiospermgenomes that were analyzed, but not in those ofgymnosperms or lower plants, implies that type 1 andtype 2 genes are ancient paralogs that diverged notonly before the split between eudicotyledons andmonocotyledons but also before the Amborella lineage(Soltis et al., 2008) diverged from the lineage that gaverise to other angiosperms. Furthermore, the presencein gymnosperms of type 1 but not type 2 genes impliesthat type 1 is the ancestral gene and type 2 arose fromit in the lineage that gave rise to angiosperms.

The existence of two similarly expressed genes inangiosperms but only one in other plants could implyeither subfunctionalization or neofunctionalization (i.e.that gene duplication was followed either by [1] sub-division of an originally broad function or [2] acqui-sition of a novel function by the duplicated gene). Theavailable data rule out neither possibility. However,assuming CoA transport to be an ancestral function(because it is shared with other eukaryotes) and thatthe type 2 gene is of quite recent origin (see above), it is

Figure 6. Expression patterns of maize genes GRMZM2G161299 andGRMZM2G420119 (abbreviated to ZM161299 and ZM420119). Rela-tive mRNA levels were determined by quantitative reverse transcription-PCR using the comparative cycle threshold method against threereference genes (FPGS, CUL, and UBCP). Error bars represent the SE

for two technical replicates of two biological replicates.

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reasonable to speculate that type 2 carriers have ac-quired a new function without losing the original one(Khersonsky and Tawfik, 2010). This new function ispresumably a capacity to transport a substrate or sub-strates other than CoA, and in this regard, it is note-worthy that the Arabidopsis peroxisomal CoA carrierPXN accepts NAD+, NADH, AMP, ADP, and adenosine39,59-phosphate as well as CoA (Agrimi et al., 2012a;Bernhardt et al., 2012). It is also noteworthy that type1 carriers restored mitochondrial CoA levels in theyeast leu5D mutant more effectively than type 2 car-riers, for this is consistent with some differentiation offunction. Therefore, it will be valuable to assess thetransport activities of type 1 and type 2 proteins inreconstituted liposomes; attempts to do this have sofar been unsuccessful.Lastly, from a practical standpoint, the conservation

of the type 1 and type 2 proteins throughout the an-giosperms, and the presence of close homologs in otherplants, means that the functional annotation “mito-chondrial CoA carrier” can be confidently propagatedfrom maize and Arabidopsis to any plant genome.

MATERIALS AND METHODS

Bioinformatics

Sequences of Arabidopsis (Arabidopsis thaliana), human, and yeast (Sac-charomyces cerevisiae) MCF carriers were taken from Palmieri et al. (2011).Maize (Zea mays) carrier sequences were from http://maizesequence.org/index.html; most other plant carrier sequences were from genomes at theNational Center for Biotechnology Information, the Joint Genome Institute(http://www.jgi.doe.gov/), or the Amborella genome database (http://www.amborella.org/). Gymnosperm sequences were based on EST contigs at theGene Index Project (http://compbio.dfci.harvard.edu/tgi/plant.html). Se-quence alignments were made with ClustalW, and phylogenetic trees wereconstructed using MEGA5 (Tamura et al., 2011). Arabidopsis gene expressionwas analyzed at Botany Array Resource (http://bar.utoronto.ca/welcome.htm; Winter et al., 2007); maize gene expression data were from qTeller (http://qteller.com/).

Constructs

All PCRs were performed using high-fidelity polymerase Phusion TAQ(New England Biolabs). Maize open reading frames GRMZM2G420119 andGRMZM2G161299 were amplified from the plasmids ZM_BFb0285D11 andZM_BFb0063E21 obtained from the Arizona Genomics Institute. Arabidopsisopen reading frames At1g14560 and At4g26180 were amplified from a leafcomplementary DNA (cDNA) library. For complementation of the yeast leu5Δstrain, coding sequences were cloned in pYES2/CT (Invitrogen). For dual-import assays, the coding sequences were cloned into pGEM-4Z (Promega)with the addition of the Kozak sequence ACC just upstream of the start codon.The plasmids (Frelin et al., 2012) used for in vivo localization studies werepRTL2ΔNS/mGFP-MCS for N-terminal GFP fusions of candidate proteins andpUC18/NcoI-mGFP for C-terminal GFP fusions. The Kozak sequence AACAwas added just upstream of the transporter sequence start codon forC-terminal GFP fusions to match that in the pRTL2ΔNS/mGFP-MCS plasmid.The primers used to build these constructs are listed in Supplemental Table S1.

Complementation of the Yeast leu5Δ Mutant

The leu5Δ deletant and the corresponding wild-type W303-1B strain weregrown in rich (YP) medium, containing 1% (w/v) yeast extract, 2% (w/v)Bacto-peptone, supplemented with 3% (w/v) glycerol. The final pH was ad-justed to 4.8 with HCl. Strains were transformed with pYES2/CT alone orpYES2/CT constructs, including positive (human SLC25A42) and negative(Arabidopsis AtNDT1 [Palmieri et al., 2009] or human SLC25A33 [Floyd et al.,

2007]) controls, as described previously (Fiermonte et al., 2009; Frelin et al.,2012).

Yeast Mitochondrial CoA Content Measurement

The leu5D strain transformed with recombinant pYES2/CT vectors wasgrown at 30°C to midlog phase in YP medium supplemented with 2% (v/v)ethanol. Mitochondria were isolated as described (Daum et al., 1982) andextracted with phenol:chloroform (1:1, v/v) for mass spectrometric analysis ofCoA. A Quattro Premier mass spectrometer interfaced with an Acquity ultra-performance liquid chromatography system (Waters) was used for liquidchromatography-tandem mass spectrometry analysis. Chromatographic res-olution was achieved using an HSS T3 column (2.1 3 100 mm, 1.8-mm particlesize; Waters). The flow rate was 0.3 mL min21. The reaction monitoringtransition selected in the positive ion mode for CoA was mass-to-charge ratio768 . 261. Calibration curves were established using standards, processedunder the same conditions as the samples, at five concentrations. The best fitwas determined using regression analysis of analyte peak area.

Dual-Import Assays

Dual-import assays with purified pea (Pisum sativum) mitochondria andchloroplasts were carried out as described (Rudhe et al., 2002; Frelin et al.,2012). The soybean (Glycine max) alternative oxidase protein GmAox (Rudheet al., 2002) and the Arabidopsis YgfZ protein At1g60990 (Waller et al., 2012)served as positive controls for mitochondrial and chloroplast import, respec-tively. Import times used in the reactions were 20 min for CoA transportercandidates and GmAox and 5 min for Arabidopsis YgfZ.

Expression and Visualization of GFP Fusion Proteins inBY-2 Cells

Expression and microscopic analyses of GFP fusion proteins in tobacco(Nicotiana tabacum) BY-2 suspension-cultured cells were performed as de-scribed previously (Frelin et al., 2012), except that endogenous mitochondrialb-ATPase protein was immunostained using mouse anti-maize mitochondrialb-ATPase E monoclonal antibodies (Luethy et al., 1993; provided by T. Elthon)and goat anti-mouse Rhodamine Red-X IgGs (Jackson ImmunoResearchLaboratories).

Analysis of Maize Gene Expression

RNA preparations were a gift from J.-C. Guan (University of Florida). Maize(inbred B73) plants were grown at the University of Florida Citra research farmin spring 2010; samples were harvested at tassel emergence. cDNAs weresynthesized with the SuperScript III First-Strand Synthesis System (LifeTechnologies) using oligo(dT)20 primer. Real-time PCR was performed usingthe MyiQ2 Real Time PCR Detection System (Bio-Rad) using iQ SYBR GreenSupermix (Bio-Rad). Amplification parameters were 95°C for 10 min followedby 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s with fluorescencemeasurement, followed by the establishment of a melting curve. Data wereanalyzed with the Bio-Rad Expression Macro using the comparative cyclethreshold method. Expression was normalized against three reference genes:FPGS (GRMZM2G393334 T01), CUL (GRMZM2G166694 T04), and UBCP(GRMZM2G102471 T01; Vandesompele et al., 2002; Manoli et al., 2012).Primers used are given in Supplemental Table S2. Primer concentrations wereoptimized for the lowest threshold cycle values, and amplification efficiencywas determined.

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers At1g14560:NP_172908; At4g26180:NP_194348;GRMZM2G420119:AFW83095; GRMZM2G161299:AFW71266; and Leu5p:NP_011865.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Alignment of candidate CoA transporters andyeast known CoA transporter.

Supplemental Figure S2. Phylogenetic analysis of candidate CoA trans-porters from Arabidopsis (At1g14560, At4g26180) and maize (ZM420119,

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ZM161299), the known CoA transporters from yeast and human, andthe complete set of MCF proteins from yeast or human.

Supplemental Figure S3. Phylogenetic analysis of candidate plant CoAtransporters and the known yeast and human CoA transporters.

Supplemental Figure S4. Complementation of growth in liquid culture ofthe yeast CoA carrier deletant leu5D by expression of candidate plantCoA carriers.

Supplemental Figure S5. Expression data for maize ZM420119 andZM161299 and Arabidopsis At1g14560 and At4g26180 in various tis-sues.

Supplemental Table S1. Primers used in this study.

Supplemental Table S2. Quantitative reverse transcription primers used inthis study.

ACKNOWLEDGMENTS

We thank M. Ziemak (University of Florida) for technical support andT. Elthon (University of Nebraska-Lincoln) for providing the anti-b-ATPaseantibodies.

Received March 19, 2013; accepted April 15, 2013; published April 16, 2013.

LITERATURE CITED

Agrimi G, Russo A, Pierri CL, Palmieri F (2012a) The peroxisomal NAD+

carrier of Arabidopsis thaliana transports coenzyme A and its derivatives.J Bioenerg Biomembr 44: 333–340

Agrimi G, Russo A, Scarcia P, Palmieri F (2012b) The human geneSLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD andNAD+. Biochem J 443: 241–247

Bairoch A (2000) The ENZYME database in 2000. Nucleic Acids Res 28:304–305

Bedhomme M, Hoffmann M, McCarthy EA, Gambonnet B, Moran RG,Rébeillé F, Ravanel S (2005) Folate metabolism in plants: an Arabi-dopsis homolog of the mammalian mitochondrial folate transportermediates folate import into chloroplasts. J Biol Chem 280: 34823–34831

Bernhardt K, Wilkinson S, Weber AP, Linka N (2012) A peroxisomalcarrier delivers NAD+ and contributes to optimal fatty acid degradationduring storage oil mobilization. Plant J 69: 1–13

Brandizzi F, Irons S, Kearns A, Hawes C (2003) BY-2 cells: culture andtransformation for live cell imaging. Curr Protoc Cell Biol 1.7.1–1.7.16

Daugherty M, Polanuyer B, Farrell M, Scholle M, Lykidis A, de Crécy-Lagard V, Osterman A (2002) Complete reconstitution of the humancoenzyme A biosynthetic pathway via comparative genomics. J BiolChem 277: 21431–21439

Daum G, Böhni PC, Schatz G (1982) Import of proteins into mitochondria:cytochrome b2 and cytochrome c peroxidase are located in the inter-membrane space of yeast mitochondria. J Biol Chem 257: 13028–13033

De Marcos Lousa C, van Roermund CW, Postis VL, Dietrich D, Kerr ID,Wanders RJ, Baldwin SA, Baker A, Theodoulou FL (2013) Intrinsicacyl-CoA thioesterase activity of a peroxisomal ATP binding cassettetransporter is required for transport and metabolism of fatty acids. ProcNatl Acad Sci USA 110: 1279–1284

Donzeau M, Káldi K, Adam A, Paschen S, Wanner G, Guiard B, BauerMF, Neupert W, Brunner M (2000) Tim23 links the inner and outermitochondrial membranes. Cell 101: 401–412

Fiermonte G, Paradies E, Todisco S, Marobbio CM, Palmieri F (2009)A novel member of solute carrier family 25 (SLC25A42) is a transporterof coenzyme A and adenosine 39,59-diphosphate in human mitochondria.J Biol Chem 284: 18152–18159

Floyd S, Favre C, Lasorsa FM, Leahy M, Trigiante G, Stroebel P, Marx A,Loughran G, O’Callaghan K, Marobbio CM, et al (2007) The insulin-like growth factor-I-mTOR signaling pathway induces the mitochon-drial pyrimidine nucleotide carrier to promote cell growth. Mol Biol Cell18: 3545–3555

Frelin O, Agrimi G, Laera VL, Castegna A, Richardson LG, Mullen RT,Lerma-Ortiz C, Palmieri F, Hanson AD (2012) Identification of mito-chondrial thiamin diphosphate carriers from Arabidopsis and maize.Funct Integr Genomics 12: 317–326

Gerdes S, Lerma-Ortiz C, Frelin O, Seaver SM, Henry CS, de Crécy-Lagard V, Hanson AD (2012) Plant B vitamin pathways and theircompartmentation: a guide for the perplexed. J Exp Bot 63: 5379–5395

Hunt MC, Alexson SE (2002) The role acyl-CoA thioesterases play in me-diating intracellular lipid metabolism. Prog Lipid Res 41: 99–130

Khersonsky O, Tawfik DS (2010) Enzyme promiscuity: a mechanistic andevolutionary perspective. Annu Rev Biochem 79: 471–505

Kirchberger S, Tjaden J, Neuhaus HE (2008) Characterization of theArabidopsis Brittle1 transport protein and impact of reduced activity onplant metabolism. Plant J 56: 51–63

Leonardi R, Zhang Y-M, Rock CO, Jackowski S (2005) Coenzyme A: backin action. Prog Lipid Res 44: 125–153

Logan DC (2006) The mitochondrial compartment. J Exp Bot 57: 1225–1243Luethy MH, Horak A, Elthon TE (1993) Monoclonal antibodies to the

a- and b-subunits of the plant mitochondrial F1-ATPase. Plant Physiol101: 931–937

Mannella CA (2006) Structure and dynamics of the mitochondrial innermembrane cristae. Biochim Biophys Acta 1763: 542–548

Manoli A, Sturaro A, Trevisan S, Quaggiotti S, Nonis A (2012) Evaluation ofcandidate reference genes for qPCR in maize. J Plant Physiol 169: 807–815

Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteinsin protoplasts of suspension cultured cells. Nat Protoc 2: 2348–2353

Neuburger M, Day DA, Douce R (1984) Transport of coenzyme A in plantmitochondria. Arch Biochem Biophys 229: 253–258

Palmieri F (2013) The mitochondrial transporter family SLC25: identifica-tion, properties and physiopathology. Mol Aspects Med 34: 465–484

Palmieri F, Pierri CL, De Grassi A, Nunes-Nesi A, Fernie AR (2011) Evo-lution, structure and function of mitochondrial carriers: a review with newinsights. Plant J 66: 161–181

Palmieri F, Rieder B, Ventrella A, Blanco E, Do PT, Nunes-Nesi A, Trauth AU,Fiermonte G, Tjaden J, Agrimi G, et al (2009) Molecular identification andfunctional characterization of Arabidopsis thaliana mitochondrial and chloro-plastic NAD+ carrier proteins. J Biol Chem 284: 31249–31259

Palmisano A, Zara V, Hönlinger A, Vozza A, Dekker PJ, Pfanner N,Palmieri F (1998) Targeting and assembly of the oxoglutarate carrier:general principles for biogenesis of carrier proteins of the mitochondrialinner membrane. Biochem J 333: 151–158

Prohl C, Pelzer W, Diekert K, Kmita H, Bedekovics T, Kispal G, Lill R(2001) The yeast mitochondrial carrier Leu5p and its human homologueGraves’ disease protein are required for accumulation of coenzyme A inthe matrix. Mol Cell Biol 21: 1089–1097

Rudhe C, Chew O, Whelan J, Glaser E (2002) A novel in vitro system forsimultaneous import of precursor proteins into mitochondria andchloroplasts. Plant J 30: 213–220

Soltis DE, Albert VA, Leebens-Mack J, Palmer JD, Wing RA,dePamphilis CW, Ma H, Carlson JE, Altman N, Kim S, et al (2008) TheAmborella genome: an evolutionary reference for plant biology. GenomeBiol 9: 402

Suppanz IE, Wurm CA, Wenzel D, Jakobs S (2009) The m-AAA proteaseprocesses cytochrome c peroxidase preferentially at the inner boundarymembrane of mitochondria. Mol Biol Cell 20: 572–580

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011)MEGA5: molecular evolutionary genetics analysis using maximumlikelihood, evolutionary distance, and maximum parsimony methods.Mol Biol Evol 28: 2731–2739

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A,Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol 3: RESEARCH0034

Waller JC, Ellens KW, Alvarez S, Loizeau K, Ravanel S, Hanson AD (2012)Mitochondrial and plastidial COG0354 proteins have folate-dependentfunctions in iron-sulphur cluster metabolism. J Exp Bot 63: 403–411

Webb ME, Smith AG (2011) Pantothenate biosynthesis in higher plants.Adv Bot Res 58: 203–255

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007)An “Electronic Fluorescent Pictograph” browser for exploring and an-alyzing large-scale biological data sets. PLoS ONE 2: e718

Wurm CA, Jakobs S (2006) Differential protein distributions define twosub-compartments of the mitochondrial inner membrane in yeast. FEBSLett 580: 5628–5634

Zara V, Ferramosca A, Robitaille-Foucher P, Palmieri F, Young JC (2009)Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70and Hsp90. Biochem J 419: 369–375

588 Plant Physiol. Vol. 162, 2013

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