Screening of the Arabidopsis thaliana genome revealed three potential homologues of mammalian and yeast mitochondrial DICs (dicarboxylate carriers) designated as DIC1, DIC2 and DIC3, each belonging to the mitochondrial carrier protein family. DIC1 and DIC2 are broadly expressed at comparable levels in all the tissues investigated. DIC1–DIC3 have been reported previously as uncoupling proteins, but direct transport assays with recombinant and reconstituted DIC proteins clearly demonstrate that their substrate specificity is unique to plants, showing the combined characteristics of the DIC and oxaloacetate carrier in yeast. Indeed, the Arabidopsis DICs transported a wide range of dicarboxylic acids including malate, oxaloacetate and succinate as well as phosphate, sulfate and thiosulfate at high rates, whereas 2-oxoglutarate was revealed to be a very poor substrate. The role of these plant mitochondrial DICs is discussed with respect to other known mitochondrial carrier family members including uncoupling proteins. It is proposed that plant DICs constitute the membrane component of several metabolic processes including the malate–oxaloacetate shuttle, the most important redox connection between the mitochondria and the cytosol.

INTRODUCTION

In addition to respiration and cellular energy supply, plant mitochondria fulfil a range of metabolic tasks, some of which are essential to several plant-specific processes such as photorespiration, C4 and CAM (crassulacean acid metabolism) photosynthesis, the utilization of carbon, nitrogen and lipid storage compounds during seed germination and the GABA (γ-aminobutyric acid) shunt. Because individual steps in these metabolic pathways are carried out in different cell compartments, there is a continuous need to move metabolites, nucleotides and cofactors into and out of the mitochondria.

The transport of solutes across the inner mitochondrial membrane is achieved by a number of proteins that belong to the MCF (mitochondrial carrier family) [15]. Family members have a tripartite structure consisting of three tandemly repeated sequences of approx. 100 amino acids in length. Each repeat contains two hydrophobic stretches that span the membrane as α-helices and a characteristic sequence motif. The yeast genome encodes 35 MCF members [6,7] and the human genome at least 50 [2]. An analysis of the Arabidopsis thaliana genome has led to the identification of 58 possible MCF members [4]. So far, several plant mitochondrial carriers have been identified by their high similarity to orthologues in other organisms [810], their transport properties upon heterologous expression and reconstitution into liposomes [1115] and by functional complementation of yeast mutants [13,16,17]. However, many other transport activities observed in plant mitochondria and/or liposomes reconstituted with plant proteins of varying purity are yet to be associated with specific protein sequences. An example is the DIC (dicarboxylate carrier), which catalyses the transport of dicarboxylates (malate and succinate) in exchange for phosphate, sulfate or thiosulfate [18]. In mammals, the DIC plays an important role in gluconeogenesis, urea synthesis and sulfur metabolism especially in the liver [19], whereas in yeast it is believed to have an anaplerotic function [20]. In plants, this carrier could play an important role in several metabolic processes including primary amino acid synthesis (ammonium assimilation), fatty acid metabolism (mobilization during seed germination), gluconeogenesis and isoprenoid biosynthesis. It may also be involved in the export of mitochondrial redox equivalents to the cytosol via the so-called ‘malate–oxaloacetate’ shuttle [21].

In the present study, we provide evidence that the gene products of At2g22500, At4g24570 and At5g09470, named DIC1, DIC2 and DIC3 respectively, are isoforms of the DIC in Arabidopsis. These proteins are 313, 313 and 337 amino acids long respectively, possess the characteristic sequence features of the MCF (see [2,4] and references therein) and display a high homology (55–70% identical amino acids and 68–81% similarity). DIC1, DIC2 and DIC3 were overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles and identified from their transport properties and kinetic characteristics as DICs. The present paper presents the first identification of three isoforms of the DIC and their genes in plants.

EXPERIMENTAL

Plant material and growth conditions

A. thaliana (ecotype Columbia) was grown in a greenhouse under either long-day (16 h light/8 h dark; at 19.5 °C/17.5 °C) or short-day (8 h light/16 h dark, at 19.5 °C/17.5 °C) conditions. Natural light was supplemented with white fluorescent light to provide 200 μmol of photons/s per m2.

Quantitative real-time PCR

Total RNA, isolated from various tissues of A. thaliana by using TRIzol® (Life Technologies), was reverse-transcribed with the GeneAmp RNA PCR Core kit (Applied Biosystems) with random hexamers as primers. The integrity of the starting RNA was verified by gel electrophoresis. Primers and probes based on the cDNA sequences of DIC1 and DIC3 were designed with Primer Express (Applied Biosystems). The forward and reverse primers corresponded to nt 1032–1050 and 1083–1102 for DIC1 and to nt 398–421 and 478–498 for DIC3. The carboxyfluorescein-Dark Quencher-labelled probes corresponded to nt 1061–1076 and 431–450 of DIC1 and DIC3 cDNAs respectively. For DIC2, the inventoried assay reagent (assay ID At02237780_s1) purchased from Applied Biosystems was used. Each 25-μl PCR mixture contained 5 μl of template (0.2 μg of reverse-transcribed first-strand cDNA), 1×ABI universal master mixture (Applied Biosystems), 900 nM of each primer and 300 nM probe for each gene. Negative controls without template were also performed. Thermal cycling was completed on a 7000 Sequence Detector System (Applied Biosystems) using the default programme of 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The PCR products were analysed by using the Sequence Detection System software 1.0 (Applied Biosystems). Quantification of the absolute number of RNA copies was performed by interpolation in standard regression curves of Ct (cycle threshold) values generated using known concentrations of DIC1, DIC2 or DIC3 cDNA [22]. The cDNAs, purified and quantified spectrophotometrically, were used to make 10-fold serial dilutions from 102 to 108 copies of plasmid DNA. The same PCR master mix and thermocycler parameters were used as described above. Our experimental conditions revealed 100 mRNA copies of DIC1, DIC2 or DIC3 with a detection rate of 100% (8 out of 8 replicates). The detection rate, however, was approx. 30% with ten mRNA molecules.

Bacterial expression and purification of DIC proteins

The coding sequences of DIC1 and DIC2 were amplified from the respective EST (expressed sequence tag) clones (AV538627 and AV541285) by PCR. The coding sequence of DIC3 was RT (reverse transcription)–PCR-amplified from flower bud RNA by using Pfu polymerase. Forward and reverse primers corresponding to the extremities of the DIC coding sequences were synthesized with additional NdeI and EcoRI sites (shown in italics) respectively (DIC1: forward 5′-GTCTTCATATGGGTCTAAAGGG-3′ and reverse 5′-CTCTTGTGAATTCAAAAGTC-3′; DIC2: forward 5′-CCACACATATGGAGTCAAAAG-3′ and reverse 5′-GAATGAATTCAAAAATCTCG-3′; and DIC3: forward 5′-CGTTCATATGGGCTTCAAACC-3′ and reverse 5′-TGAAGAATTCTGGGAATCTCAA-3′). The PCR products were sequenced and cloned into the pMW7 expression vector. The sequences of the inserts were verified using the BigDye™ Terminator kit (PerkinElmer) according to the manufacturer's instructions and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The constructs were used to transform E. coli C0214(DE3) cells, and the recombinant DIC proteins were overproduced as inclusion bodies at 37 °C, as described previously [23]. Inclusion bodies were purified on a sucrose density gradient, washed at 4 °C with TE buffer (10 mM Tris/HCl and 1 mM EDTA, pH 7), then twice with a buffer containing Triton X-114 (3%, w/v), 1 mM EDTA and 10 mM Pipes/NaOH (pH 7.0) and once again with TE buffer [23]. DIC proteins were solubilized in 1.7% (w/v) sarkosyl, 0.1 mM EDTA and 10 mM Tris/HCl (pH 7.0). Small insoluble residues were removed by centrifugation at 258000 g for 1 h at 4 °C.

Reconstitution into liposomes and transport measurements

The recombinant proteins in sarkosyl were reconstituted into liposomes in the presence of substrates, as described previously [24]. External substrate was removed from proteoliposomes on Sephadex G-75 columns (Amersham Biosciences). Transport at 25 °C was started by adding [35S]sulfate, [14C]malate or [33P]Pi (Amersham) to the proteoliposomes and terminated by addition of 30 mM pyridoxal 5′-phosphate and 10 mM bathophenanthroline (the ‘inhibitor-stop’ method [24]). In controls, the inhibitors were added with the labelled substrate. The transport measurements were carried out at the same internal and external pH value of 7.0. External radioactivity was removed from quenched samples on Sephadex G-75 columns, and internal radioactivity was measured [24]. The experimental values were corrected by subtracting control values. The initial transport rate was calculated from the radioactivity taken up by proteoliposomes after 20 s (in the initial linear range of substrate uptake).

Other methods

Proteins were separated by SDS/PAGE and stained with Coomassie Blue. The N-termini were sequenced and the amount of purified proteins was estimated by laser densitometry of stained samples by using carbonic anhydrase as a protein standard [25]. The amount of protein incorporated into liposomes was measured, as described previously [25]; in all cases, it was approx. 25% of the protein added to the reconstitution mixture.

RESULTS

Isolation and characterization of DIC cDNAs

The protein sequence of the human DIC, encoded by the SLC25A10 gene [2,26], was used to search databases for homologous plant sequences. Several related ESTs were identified that corresponded to the A. thaliana genes At2g22500 and At4g24570. The cDNAs corresponding to the EST database entries AV538627 and AV541285 were retrieved from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan) and sequenced. Their complete nucleotide sequences corresponded to ‘full-length’ cDNAs [including 5′-UTR (5′-untranslated region) and 3′-UTR sequences]. One of these cDNAs contained a 942-bp ORF (open reading frame) that encoded a polypeptide of 313 amino acids (referred to as DIC1 hereinafter) with a calculated molecular mass of 33.4 kDa (Figure 1). Analysis of the other cDNA revealed a 942-bp ORF encoding a 313-amino-acid polypeptide (DIC2) with a calculated molecular mass of 32.9 kDa (Figure 1). By screening Arabidopsis databases, we identified another gene (At5g09470) that was also related to the human DIC but had no reported ESTs. The absence of ESTs for At5g09470 raised the question of whether it was a pseudogene. However, PCR experiments using different cDNA libraries indicated that its RNA was present in flower buds and siliques (results not shown). Therefore the coding region of this gene was RT–PCR-amplified from flower bud RNA by using primers based on the genomic sequence of At5g09470. The deduced protein sequence of DIC3 consisted of 337 amino acids with a molecular mass of 36.1 kDa (Figure 1). DIC1 and DIC2 share 70% identical amino acids, whereas the protein sequence of DIC3 is 55–60% identical with those of the other two Arabidopsis DICs. The major difference between DIC3 and the other two DIC proteins is the presence of several small insertions in a segment of DIC3 located after the first 36 N-terminal residues (Figure 1). Therefore DIC1 and DIC2 are more closely related, as is also indicated by the fact that their genes contain no introns, whereas the DIC3 gene possesses a canonical type II intron spanning the 428–686 nt region of the At5g09470 sequence. All three Arabidopsis DIC proteins belong to the MCF given their tripartite structure, the presence of two hydrophobic regions in each repeat and the 3-fold repetition of the signature motif (Figure 1).

Sequence alignment of the three Arabidopsis DIC proteins

Figure 1
Sequence alignment of the three Arabidopsis DIC proteins

The deduced amino acid sequences of the DIC genes were aligned using ClustalX, with identical residues indicated by black boxes. Added gaps are indicated by hyphens. Continuous lines were placed below the conserved motif that is characteristic of the mitochondrial carrier protein family.

Figure 1
Sequence alignment of the three Arabidopsis DIC proteins

The deduced amino acid sequences of the DIC genes were aligned using ClustalX, with identical residues indicated by black boxes. Added gaps are indicated by hyphens. Continuous lines were placed below the conserved motif that is characteristic of the mitochondrial carrier protein family.

Molecular and phylogenetic relationship of Arabidopsis DIC proteins to other mitochondrial transporters

Amino acid sequence comparisons revealed that Arabidopsis DICs showed the highest degree of similarity to other plant proteins that had been annotated as putative (oxo)dicarboxylate carriers in databases. For example, DIC1 exhibited 73% identity with a putative DIC from Trifolium pratense (BAE71294), 68% identity with a putative carrier protein from Oryza sativa (EAZ07364), 67% identity with a putative uncoupling protein from Saccharum officinarum (AAU11466) and 60% identity with a 2-oxoglutarate carrier-like protein from O. sativa (BAD17507). Among mitochondrial transporters from other organisms, Arabidopsis DICs showed the highest homology with the already identified mitochondrial DICs, sharing 42% identical amino acids with the human DIC [26] and 37% with the Caenorhabditis elegans DIC [27]. Furthermore, the MCF member in Saccharomyces cerevisiae that is most closely related to the Arabidopsis DICs was the yeast DIC [6], although with only 32% identity. A phylogenetic analysis (Figure 2) carried out using the Arabidopsis DIC sequences and other mitochondrial carriers for di- and tri-carboxylates as well as those of uncoupling proteins revealed that DIC1, DIC2 and DIC3 are monophyletic and form a group close to those of non-plant DICs, the plant DTCs (di- and tri-carboxylate carriers) and animal oxoglutarate carriers. However, plant and animal DICs formed two distinct clusters, with the yeast DIC belonging to the latter. In addition, the high degree of homology between DIC1, DIC2 and DIC3 strongly suggested that they may be isoforms of the same transporter.

Phylogenic tree of amino acid sequences of mitochondrial transporters from various organisms

Figure 2
Phylogenic tree of amino acid sequences of mitochondrial transporters from various organisms

The unrooted dendrogram originated from an alignment performed with ClustalX (1.75) software by using default parameters and visualized by using Phylodendron TreePrint (http://www.es.embnet.org/Doc/phylodendron/treeprint-form.html). Branch lengths are drawn proportional to the amount of sequence change. The bar indicates the number of substitutions per residue, with 0.1 corresponding to a distance of 10 substitutions per 100 residues. The proteins have the following accession numbers: yeast DIC, U79459; C. elegans DIC, X76114; human DIC, AJ131613; mouse DIC, AF188712; rat DIC, AJ223355; millet DTC, D45073; At-DTC (A. thaliana DTC), AJ311780; tobacco DTC, Q8SF04; C. elegans OGC, AAB37890; bovine OGC, M60662; rat OGC, U84727; human OGC, X66114; human UCP5, O95258; AtPUMP3, F7A19_22; human UCP4, O95847; AtPUMP1, CAA11757; AtPUMP2, NP_568894; rat UCP1, P04633; human UCP1, P25874; human UCP2, P55851; rat UCP2, P56500; rat UCP3, P56499; human UCP3, P55916; yeast OAC, P32332; At4g03115, NP_680566; At-SFC, 15450697; yeast SFC, Z49595; yeast CIC, U17503; C. elegans CIC, P34519; bovine CIC, P79110; rat CIC, P32089; human CIC, BC004980; At-DIC3, AM236861; At-DIC1, AM236862; and At-DIC2, AM236863. Abbreviations: At, A. thaliana; OGC, oxoglutarate carrier; PUMP and UCP, uncoupling proteins; OAC, oxaloacetate carrier; SFC, succinate/fumarate carrier; CIC, citrate carrier.

Figure 2
Phylogenic tree of amino acid sequences of mitochondrial transporters from various organisms

The unrooted dendrogram originated from an alignment performed with ClustalX (1.75) software by using default parameters and visualized by using Phylodendron TreePrint (http://www.es.embnet.org/Doc/phylodendron/treeprint-form.html). Branch lengths are drawn proportional to the amount of sequence change. The bar indicates the number of substitutions per residue, with 0.1 corresponding to a distance of 10 substitutions per 100 residues. The proteins have the following accession numbers: yeast DIC, U79459; C. elegans DIC, X76114; human DIC, AJ131613; mouse DIC, AF188712; rat DIC, AJ223355; millet DTC, D45073; At-DTC (A. thaliana DTC), AJ311780; tobacco DTC, Q8SF04; C. elegans OGC, AAB37890; bovine OGC, M60662; rat OGC, U84727; human OGC, X66114; human UCP5, O95258; AtPUMP3, F7A19_22; human UCP4, O95847; AtPUMP1, CAA11757; AtPUMP2, NP_568894; rat UCP1, P04633; human UCP1, P25874; human UCP2, P55851; rat UCP2, P56500; rat UCP3, P56499; human UCP3, P55916; yeast OAC, P32332; At4g03115, NP_680566; At-SFC, 15450697; yeast SFC, Z49595; yeast CIC, U17503; C. elegans CIC, P34519; bovine CIC, P79110; rat CIC, P32089; human CIC, BC004980; At-DIC3, AM236861; At-DIC1, AM236862; and At-DIC2, AM236863. Abbreviations: At, A. thaliana; OGC, oxoglutarate carrier; PUMP and UCP, uncoupling proteins; OAC, oxaloacetate carrier; SFC, succinate/fumarate carrier; CIC, citrate carrier.

Expression of DICs in plant organs

The TAIR (The Arabidopsis Information Resource) database indicates that DIC1 (108 ESTs) and DIC2 (70 ESTs) are more highly expressed than DIC3 (0 ESTs). The expression of the DIC isoforms in A. thaliana was examined in different tissues by quantitative real-time PCR. To determine the absolute number of RNA copies of DIC1, DIC2 and DIC3, standard calibration curves were constructed using 102–108 copies of their cDNAs. Under our experimental conditions, the threshold of 100 copies of each gene was detected with accuracy and reproducibility. From the fluorescence signals obtained with the different tissue samples interpolated in the calibration curves, the number of RNA copies was calculated to vary between 67583 and 281397 per μg of total RNA for DIC1 and between 71176 and 224582 per μg of total RNA for DIC2 (Figure 3). DIC1 was more expressed in root and stem than in flowers, leaf and seedling, whereas DIC2 more in stem, root and leaf than in seedling and flowers. The number of mRNA copies of DIC3 in the investigated tissues was below the detection limit of 500 copies per μg of total RNA. These observations are in good agreement with the Arabidopsis gene expression data in public microarray data collections (available at Genevestigator: https://www.genevestigator.ethz.ch/) [28], which show that DIC1 and DIC2 are expressed in all the tissues at comparable levels and that the expression of DIC3 is very low or absent.

Expression of Arabidopsis DIC in various tissues

Figure 3
Expression of Arabidopsis DIC in various tissues

Quantitative real-time PCR experiments of DIC1 (grey bars) and DIC2 (black bars) were conducted on cDNAs prepared by RT of total RNAs from the indicated tissues by using specific primers and probes based on Arabidopsis DIC. The values in number of copies/μg of total RNA are the means±S.E.M. for four independent experiments.

Figure 3
Expression of Arabidopsis DIC in various tissues

Quantitative real-time PCR experiments of DIC1 (grey bars) and DIC2 (black bars) were conducted on cDNAs prepared by RT of total RNAs from the indicated tissues by using specific primers and probes based on Arabidopsis DIC. The values in number of copies/μg of total RNA are the means±S.E.M. for four independent experiments.

Bacterial expression and functional characterization of recombinant DIC proteins

DIC1, DIC2 and DIC3 were expressed at high levels in E. coli C0214(DE3). They accumulated as inclusion bodies and were purified by centrifugation and washing (Figure 4, lanes 3, 6 and 9). The identity of the purified proteins was confirmed by N-terminal sequencing. Approx. 70–100 mg of each purified protein per litre of culture was obtained. The proteins were not detected in the cells harvested after induction of expression but lacking the coding sequence in the vector (Figure 4, lanes 1, 4 and 7) nor in bacteria harvested immediately before induction (results not shown).

Expression in E. coli and purification of Arabidopsis DIC proteins

Figure 4
Expression in E. coli and purification of Arabidopsis DIC proteins

Proteins were separated by SDS/12.5%-PAGE and stained with Coomassie Brilliant Blue dye. Indicated molecular-mass standards on the left-hand side correspond, from top to bottom to BSA, ovotransferrin, carbonic anhydrase and cytochrome c; lanes 1 and 2, E. coli C0214(DE3) containing the expression vector with (lane 2) and without (lane 1) the DIC1 coding sequence; lanes 4 and 5, E. coli C0214(DE3) containing the expression vector with (lane 5) and without (lane 4) the DIC2 coding sequence; lanes 7 and 8, E. coli C0214(DE3) containing the expression vector with (lane 8) and without (lane 7) the DIC3 coding sequence; lanes 3, 6 and 9, purified DIC1, DIC2 and DIC3 originating from bacteria shown in lanes 2, 5 and 8 respectively. Samples were taken 5 h after induction. The same number of bacteria was analysed in each sample.

Figure 4
Expression in E. coli and purification of Arabidopsis DIC proteins

Proteins were separated by SDS/12.5%-PAGE and stained with Coomassie Brilliant Blue dye. Indicated molecular-mass standards on the left-hand side correspond, from top to bottom to BSA, ovotransferrin, carbonic anhydrase and cytochrome c; lanes 1 and 2, E. coli C0214(DE3) containing the expression vector with (lane 2) and without (lane 1) the DIC1 coding sequence; lanes 4 and 5, E. coli C0214(DE3) containing the expression vector with (lane 5) and without (lane 4) the DIC2 coding sequence; lanes 7 and 8, E. coli C0214(DE3) containing the expression vector with (lane 8) and without (lane 7) the DIC3 coding sequence; lanes 3, 6 and 9, purified DIC1, DIC2 and DIC3 originating from bacteria shown in lanes 2, 5 and 8 respectively. Samples were taken 5 h after induction. The same number of bacteria was analysed in each sample.

Each recombinant protein was reconstituted into liposomes and their transport activities for potential substrates were tested in homo-exchange experiments (i.e. with the same substrate inside and outside). In the reconstituted system, DIC1, DIC2 or DIC3 catalysed an active [35S]sulfate/sulfate exchange that was completely inhibited by a mixture of pyridoxal 5′-phosphate and bathophenanthroline. They did not catalyse homo-exchanges for pyruvate, citrate, carnitine, glutamate and ATP (internal concentration, 10 mM; external concentration, 1 mM) (results not shown). Importantly, no [35S]sulfate/sulfate exchange activity was detected if the protein had been boiled before incorporation into liposomes or if proteoliposomes were reconstituted with sarkosyl-solubilized material from bacterial cells either lacking the expression vector for DIC or harvested immediately before induction of expression. Likewise, no such activity was detected in liposomes reconstituted with three unrelated mitochondrial carriers, Ort1p [29], Ggc1p [30] and Sam5p [31], which had been expressed and purified from E. coli CO214(DE3).

As shown in Figure 5, the uptake of [35S]sulfate by sulfate/sulfate exchange, catalysed by DIC2, followed a first-order kinetics (rate constant, 0.41 min−1; initial rate, 327 μmol/min per g of protein) with isotopic equilibrium being reached exponentially. In contrast, no [35S]sulfate uptake was observed without internal substrate (Figure 5), demonstrating that DIC2 does not catalyse unidirectional transport (uniport) but only an exchange of substrates. Similar results were obtained with reconstituted DIC1 and DIC3.

Kinetics of [35S]sulfate transport in proteoliposomes reconstituted with DIC2

Figure 5
Kinetics of [35S]sulfate transport in proteoliposomes reconstituted with DIC2

[35S] sulfate (0.1 mM) was added to proteoliposomes containing 10 mM sulfate (exchange; ●) or 5 mM NaCl and no substrate (uniport; ○). Similar results were obtained in three independent experiments.

Figure 5
Kinetics of [35S]sulfate transport in proteoliposomes reconstituted with DIC2

[35S] sulfate (0.1 mM) was added to proteoliposomes containing 10 mM sulfate (exchange; ●) or 5 mM NaCl and no substrate (uniport; ○). Similar results were obtained in three independent experiments.

The substrate specificity of the three reconstituted A. thaliana DIC proteins was investigated in detail by measuring the uptake of [35S]sulfate into proteoliposomes that had been preloaded with a variety of potential substrates. DIC1 and DIC2 allowed [35S]sulfate to be exchanged with internal sulfate, phosphate, arsenate, thiosulfate, malate, malonate, succinate, oxaloacetate, maleate or oxalate (Figure 6). DIC3 yielded higher activities of [35S]sulfate uptake with internal sulfate, thiosulfate, oxaloacetate and oxalate than with phosphate, arsenate, malate, malonate, succinate and maleate (Figure 6). Each of the three DICs exhibited very low transport activity with internal sulfite and 2-oxoglutarate, whereas virtually no transport was detected with internal fumarate, aspartate, citrate, cis-aconitate (Figure 6), and arginine, ATP, carnitine, pyruvate, glutamate, glutamine or glutathione (results not shown). Notably, in liposomes reconstituted with DIC1 and DIC2, [35S]sulfate exchanged better with sulfate than with thiosulfate and with malonate rather than with oxalate, whereas in liposomes reconstituted with DIC3, [35S]sulfate exchanged better with oxalate than with malonate.

Substrate specificity of DIC1, DIC2 and DIC3

Figure 6
Substrate specificity of DIC1, DIC2 and DIC3

Liposomes, reconstituted with DIC1 in (A), DIC2 in (B) and DIC3 in (C), were preloaded internally with various substrates (concentration 20 mM). Transport was started by the addition of 0.1 mM [35S]sulfate and terminated after 20 s. The values are the means±S.E.M. for at least three independent experiments.

Figure 6
Substrate specificity of DIC1, DIC2 and DIC3

Liposomes, reconstituted with DIC1 in (A), DIC2 in (B) and DIC3 in (C), were preloaded internally with various substrates (concentration 20 mM). Transport was started by the addition of 0.1 mM [35S]sulfate and terminated after 20 s. The values are the means±S.E.M. for at least three independent experiments.

The [35S]sulfate/sulfate exchange reaction catalysed by the three Arabidopsis DIC proteins was inhibited strongly by pyridoxal 5′-phosphate, bathophenanthroline, Bromocresol Purple and mercuric chloride (Figure 7). The organic mercurials, mersalyl and p-hydroxymercuribenzoate, and the impermeable dicarboxylate analogues, butylmalonate and phenylsuccinate, markedly inhibited the activity of DIC1 and DIC2 and, to a lesser extent, that of DIC3. In contrast, inhibitors of other characterized mitochondrial carriers, such as N-ethylmaleimide, α-cyano-4-hydroxycinnamate, carboxyatractyloside and bongkrekic acid (Figure 7), and 1,2,3-benzenetricarboxylate (results not shown), had very little effect on the activity of the Arabidopsis DIC proteins.

Effect of inhibitors on the [35S]sulfate/sulfate exchange by DIC1, DIC2 and DIC3

Figure 7
Effect of inhibitors on the [35S]sulfate/sulfate exchange by DIC1, DIC2 and DIC3

Proteoliposomes were preloaded internally with 20 mM sulfate and transport was initiated by adding 0.1 mM [35S]sulfate to proteoliposomes reconstituted with DIC1 (white bars), DIC2 (grey bars) or DIC3 (black bars). The incubation time was 20 s. Thiol reagents and α-cyano-4-hydroxycinnamate were added 2 min before the labelled substrate; the other inhibitors were added together with [35S]sulfate. The final concentrations of the inhibitors were 0.1 mM for mersalyl (MER), p-hydroxymercuribenzoate (HMB) and mercuric chloride (HgCl2); 2 mM for pyridoxal 5-phosphate (PLP), bathophenanthroline (BAT), N-ethylmaleimide (NEM), butylmalonate (BMA) and phenylsuccinate (PHS); 0.3 mM for Bromocresol Purple (BCP); 10 μM for carboxyatractyloside (CAT) and bongkrekic acid (BKA); 20 μM for α-cyano-4-hydroxycinnamate (CCN); and 0.2% for tannic acid (TAN). The extent of inhibition (percentage) for each carrier from a representative experiment is reported. Similar results were obtained in at least three experiments.

Figure 7
Effect of inhibitors on the [35S]sulfate/sulfate exchange by DIC1, DIC2 and DIC3

Proteoliposomes were preloaded internally with 20 mM sulfate and transport was initiated by adding 0.1 mM [35S]sulfate to proteoliposomes reconstituted with DIC1 (white bars), DIC2 (grey bars) or DIC3 (black bars). The incubation time was 20 s. Thiol reagents and α-cyano-4-hydroxycinnamate were added 2 min before the labelled substrate; the other inhibitors were added together with [35S]sulfate. The final concentrations of the inhibitors were 0.1 mM for mersalyl (MER), p-hydroxymercuribenzoate (HMB) and mercuric chloride (HgCl2); 2 mM for pyridoxal 5-phosphate (PLP), bathophenanthroline (BAT), N-ethylmaleimide (NEM), butylmalonate (BMA) and phenylsuccinate (PHS); 0.3 mM for Bromocresol Purple (BCP); 10 μM for carboxyatractyloside (CAT) and bongkrekic acid (BKA); 20 μM for α-cyano-4-hydroxycinnamate (CCN); and 0.2% for tannic acid (TAN). The extent of inhibition (percentage) for each carrier from a representative experiment is reported. Similar results were obtained in at least three experiments.

Kinetic characteristics of the recombinant DIC proteins

The kinetic constants of the recombinant purified Arabidopsis DIC proteins were determined by measuring the initial transport rate at various external [35S]sulfate, [14C]malate or [33P]Pi concentrations in the presence of a constant saturating internal concentration of the same substrate (homo-exchange). The mean Km and Vmax values and their S.E.M. values are shown in Table 1. For each DIC protein, the specific activity (Vmax) was not significantly different for the three substrates tested. However, DIC3 displayed a Vmax value that was approximately one order of magnitude higher than that of DIC1 and 2-fold higher than that of DIC2. The apparent transport affinity (Km) of all three DIC proteins for sulfate was lower than the Km values for phosphate and malate, which were very similar to each other. In the case of DIC3, the Km for sulfate was one order of magnitude lower than the Km values for phosphate and malate. In addition, the Km of DIC3 for sulfate was 3–4-fold lower than the Km values of DIC1 and DIC2 for the same substrate.

Table 1
Kinetic constants of substrate uptake into proteoliposomes reconstituted with recombinant DIC1, DIC2 or DIC3

Data were obtained from Lineweaver–Burk plots of the rates of the homo-exchanges (the same substrate inside and outside) under variation of the external substrate concentration. The concentration of the internal substrate was 20 mM. The values given in the Table are the means±S.E.M. for at least four independent experiments.

Carrier Substrate Km (mM) Vmax (mmol/min per g of protein) 
DIC1 Sulfate 0.23±0.06 0.19±0.05 
 Malate 0.40±0.09 0.29±0.06 
 Phosphate 0.47±0.06 0.22±0.05 
DIC2 Sulfate 0.17±0.04 0.82±0.17 
 Malate 0.51±0.07 1.01±0.11 
 Phosphate 0.66±0.07 0.81±0.15 
DIC3 Sulfate 0.06±0.02 1.93±0.24 
 Malate 0.79±0.05 2.21±0.31 
 Phosphate 0.69±0.04 1.78±0.16 
Carrier Substrate Km (mM) Vmax (mmol/min per g of protein) 
DIC1 Sulfate 0.23±0.06 0.19±0.05 
 Malate 0.40±0.09 0.29±0.06 
 Phosphate 0.47±0.06 0.22±0.05 
DIC2 Sulfate 0.17±0.04 0.82±0.17 
 Malate 0.51±0.07 1.01±0.11 
 Phosphate 0.66±0.07 0.81±0.15 
DIC3 Sulfate 0.06±0.02 1.93±0.24 
 Malate 0.79±0.05 2.21±0.31 
 Phosphate 0.69±0.04 1.78±0.16 

DISCUSSION

In plants as well as in animals, knowledge concerning the mitochondrial DIC has existed for a long time, and its main properties have been studied in isolated intact mitochondria (for reviews, see [1,3] and references therein). However, in plants, the protein(s) responsible for the DIC has not been identified to date. The results reported in the present study demonstrate that we have identified DIC1, DIC2 and DIC3 as isoforms of the DIC in A. thaliana, because the properties of the recombinant and reconstituted proteins closely resemble those of the native plant and animal DICs in substrate specificity, transport affinities and inhibitor-sensitivity.

Similar to the biochemically characterized DICs from non-plant organisms, DIC1, DIC2 and DIC3 transport malate, oxaloacetate, succinate, maleate, malonate, phosphate, sulfate and thiosulfate. The substrate specificity of the Arabidopsis DIC proteins is distinct from any other mitochondrial carrier of known function, including the related oxoglutarate [32], succinate/fumarate [33], oxaloacetate [34] and oxodicarboxylate [35] carriers (whose principal substrates are 2-oxoglutarate and malate; succinate and fumarate; oxaloacetate and sulfate; and oxoadipate and oxoglutarate respectively). It also partially differs from that of mammalian and yeast DICs, which are their closest sequence homologues. Thus Arabidopsis DICs transport oxaloacetate, sulfate and thiosulfate more efficiently than animal DICs as well as oxoglutarate, although to a very low extent. Furthermore, compared with rat, C. elegans and yeast DICs, the plant carriers show similar Km values for malate (0.4–0.79 mM) but lower Km values for phosphate (0.47–0.69 mM versus 1.41–1.77 mM). These differences are probably accounted for by the phylogenic analysis reported in Figure 2, since Arabidopsis DICs form a separate clade with respect to fungal and animal DICs. Plants, yeast and mammals appear to have evolved different types of mitochondrial carriers for di- and tri-carboxylic acids: the dicarboxylate, oxoglutarate, oxoadipate/oxoglutarate and citrate carriers in mammals; the dicarboxylate, oxaloacetate, succinate/fumarate, oxoadipate/oxoglutarate and citrate carriers in yeast; and the dicarboxylate, succinate/fumarate and dicarboxylate–tricarboxylate carriers in plants. Interestingly, plants appear to contain ‘hybrid’ carriers when compared with yeast and mammals: their DTC possesses the combined characteristics of the mammalian oxoglutarate and citrate carriers [12] and their DIC exhibits the combined characteristics of the yeast dicarboxylate and oxaloacetate carriers (the present study).

Although the Arabidopsis DIC isoforms have many properties in common, they differ in some respects. Relative to sulfate transport, dicarboxylates, phosphate and arsenate are transported more efficiently by DIC1 and DIC2 than by DIC3, whereas thiosulfate and oxaloacetate are more efficiently transported by DIC3 when compared with DIC1 and DIC2. Furthermore, DIC3 is inhibited considerably less by butylmalonate and phenylsuccinate, as well as by the organic mercurials, with respect to DIC1 and DIC2. These differences are consistent with the fact that DIC1 and DIC2 share 70% identical amino acids, whereas DIC3 shares only 55–60% identical amino acids with DIC1 and DIC2. Indeed, DIC3 seems to exhibit a substrate specificity that resembles more the yeast oxaloacetate carrier.

In a series of studies, DIC1, DIC2 and DIC3 have been reported to be mitochondrial uncoupling proteins named AtPUMP4–6 (where PUMP is plant uncoupling mitochondrial protein) [3638]. This conclusion was based on sequence homologies between AtPUMP1–3 and DIC1, DIC2 and DIC3 [3638] and on the observation that recombinant AtPUMP6 (i.e. DIC3) mediated a linoleic acid-stimulated H+ flux in reconstituted liposomes [37]. However, DIC1, DIC2 and DIC3 are phylogenetically closer to animal and yeast DICs than to uncoupling proteins including AtPUMP1–3 (Figure 2); recombinant and purified DIC1, DIC2 and DIC3 all transport the characteristic substrates of the DIC by an exchange mechanism (the present study). Linoleic acid may increase the permeability of the proteoliposomes in a non-DIC3-specific manner; the Vmax of the reported H+ flux [37] is approximately an order of magnitude lower than the Vmax of dicarboxylate transport by DIC3 (the present study). Furthermore, even if solid evidence for a DIC3-mediated H+ flux were provided, this finding alone would not be enough to identify DIC3 as an uncoupling protein instead of a DIC, as our phylogenetic analysis (Figure 2) suggests and direct metabolite transport assays (Figure 6) demonstrate. In fact, it has been reported that the DIC is involved in the protonophoric action of fatty acids in mitochondria [39]. Moreover, a carrier-dependent fatty acid-stimulated flux of H+ has often been observed in mitochondria and liposomes reconstituted with mitochondrial carriers [4042].

Expression analyses of the Arabidopsis DIC genes show that DIC1 and DIC2 are present in all of the plant tissues/organs analysed, implying that DIC plays an essential role in plant cell metabolism. However, a functional redundancy may exist between the DIC isoforms as well as between the DIC and other similar plant MCF members such as the DTC and succinate/fumarate carrier. Our results further show that DIC1 and DIC2 exhibit comparable levels of expression. In addition, under certain stress conditions, subtle differences emerge in DIC isoform expression. DIC1 expression is reduced under anoxia and hypoxia, whereas DIC2 expression is reduced by UV treatment. Conversely, DIC1 expression is increased by salt stress and DIC2 by treatment with H2O2 (results not shown). Such differences could suggest that these DIC isoforms might have specific functions, perhaps in specific plant tissues/organs.

Because the DICs transport a broad spectrum of dicarboxylates, they may potentially play a role in a number of important metabolic functions that require organic acid flux to or from the mitochondria. First, it is very likely that DIC plays an anaplerotic role, leading to the transport of dicarboxylic acids into the mitochondria (in exchange for phosphate or sulfate), which are then used as respiratory substrates. This function is further supported by the early findings that malate and succinate oxidation by plant mitochondria is inhibited by DIC inhibitors [43,44]. Secondly, the malate/oxaloacetate exchange catalysed by DIC, coupled with the cytosolic and mitochondrial NAD-dependent malate dehydrogenase activities, would allow the transfer of reducing equivalents (i.e. NADH) from the mitochondrial matrix to the cytosol (and vice versa). Redox shuttles are important in photosynthetic organs such as the leaves of C3 plants. The chloroplastic malate valve (see [45]) and mitochondrial malate/oxaloacetate shuttle are involved in the light-dependent photorespiratory cycle that requires malate to be shifted to the peroxisomes for the production of NADH in the synthesis of glycerate from hydroxypyruvate [46,47]. Indeed, it was shown that mitochondria can produce 25% of the required reducing power for photorespiration via the malate/oxaloacetate shuttle [46]. It had been reported that the mitochondrial redox shuttle was composed of two electrogenic uniporters linked for the sake of charge compensation [48]. However, this interpretation arose from artificial results due to high metabolite concentrations; a single malate/oxaloacetate antiporter is believed to carry out this function [21]. The DIC proteins described in the present study possess the characteristics of the proposed malate shuttle. It was suggested that under normal cytosolic NADH/NAD levels, this carrier would only export reducing equivalents from the mitochondria [46]. More recently, it has been shown that the shuttle can be reversed and that the malate/oxaloacetate exchange across the inner mitochondrial membrane is the rate-limiting step in the import of redox equivalents to wheat and potato mitochondria [49]. The authors proposed the existence of a novel malate/oxaloacetate antiporter but could not prove it at the molecular level. When this mitochondrial carrier is coupled with the chloroplastic malate valve, the plant acquires the ability to balance the cellular energy supply and control redox poise [45]. Furthermore, shifting malate allows the production of NADH in the cytosol to fuel nitrate reductase, an important NADH-consuming enzyme that reduces nitrate to nitrite. In addition, the high expression of DIC1 and DIC2 in the cotyledons, as deduced from the public microarray data, and their capacity to transport succinate and malate, suggest that they can play a role in the mobilization of storage lipids during germination and/or in gluconeogenesis.

The molecular identification of plant DICs paves the way to the evaluation of their individual role(s) within the mitochondrial membrane under different physiological conditions. This will require the isolation and characterization of mutants for each DIC isoform and perhaps the study of double or even triple mutants.

N. P. was supported by a Ph.D. grant from the French Ministry of National Education, Research and Technology, and M. H. was funded by the CNRS and the Université Paris Sud-XI. Research in the laboratories of F. P and L. P. was supported by grants from MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica) [MIUR-PRIN (progetti di ricerca di interesse nazionale) and FIRB (Fondi Istituzionali per la Ricerca di Base)], CNR (Consiglio Nazionale delle Ricerche) and CEGBA (Centro di Eccellenza Geni in campo Biosanitaro ed Agroalimentare). We thank the Kazusa DNA Research Institute (Chiba, Japan) for sending us the EST clones.

Abbreviations

     
  • DIC

    dicarboxylate carrier

  •  
  • DTC

    di- and tri-carboxylate carrier

  •  
  • EST

    expressed sequence tag

  •  
  • MCF

    mitochondrial carrier family

  •  
  • ORF

    open reading frame

  •  
  • PUMP

    plant uncoupling mitochondrial protein

  •  
  • RT

    reverse transcription

  •  
  • UTR

    untranslated region

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Author notes

1

Present address: Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS-IRD, Université de Perpignan, Perpignan 66860, France.

The sequence data reported in this paper as DIC1, DIC2 and DIC3 have been deposited in the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases with accession numbers AM236862, AM236863 and AM236861 respectively.