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J. Biol. Chem., Vol. 277, Issue 27, 24204-24211, July 5, 2002

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Identification of a Novel Transporter for Dicarboxylates and Tricarboxylates in Plant Mitochondria

BACTERIAL EXPRESSION, RECONSTITUTION, FUNCTIONAL CHARACTERIZATION, AND TISSUE DISTRIBUTION^*

Nathalie Picault^§¶, Luigi Palmieri^§, Isabella Pisano, Michael Hodges, and Ferdinando Palmieri^**

From the  Institut de Biotechnologie des Plantes, CNRS UMR8618, Université de Paris Sud, 91405 Orsay, Cedex, France and the  Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy

Received for publication, March 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A cDNA from Arabidopsis thaliana and four related cDNAs from Nicotiana tabacum that we have isolated encode hitherto unidentified members of the mitochondrial carrier family. These proteins have been overexpressed in bacteria and reconstituted into phospholipid vesicles. Their transport properties demonstrate that they are orthologs/isoforms of a novel mitochondrial carrier capable of transporting both dicarboxylates (such as malate, oxaloacetate, oxoglutarate, and maleate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate). The newly identified dicarboxylate-tricarboxylate carrier accepts only the single protonated form of citrate (H-citrate²) and the unprotonated form of malate (malate²) and catalyzes obligatory, electroneutral exchanges. Oxoglutarate, citrate, and malate are mutually competitive inhibitors, showing K_i close to the respective K_m. The carrier is expressed in all plant tissues examined and is largely spread in the plant kingdom. Furthermore, nitrate supply to nitrogen-starved tobacco plants leads to an increase in its mRNA in roots and leaves. The dicarboxylate-tricarboxylate carrier may play a role in important plant metabolic functions requiring organic acid flux to or from the mitochondria, such as nitrogen assimilation, export of reducing equivalents from the mitochondria, and fatty acid elongation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transport of metabolites in and out of mitochondria is mediated by a family of related carrier proteins that span the lipid bilayer of the inner membrane (1). The polypeptide sequences of members of this family are made up of three related domains of about 100 amino acids repeated in tandem, each probably being folded into two trans-membrane -helices connected by an extensive hydrophilic loop. The repeats in the various family members are all related, and various sequence features are conserved (1, 2). So far 14 mitochondrial carriers have been identified and sequenced from yeast and animals (see Ref. 3 and references therein). The functions of many other family members found in genomic sequences are unknown.

In plants, many transport activities observed in mitochondria and/or in liposomes reconstituted with more or less purified protein fractions await to be associated with specific protein sequences (for a review see Ref. 4). These include the transport of specific dicarboxylates and tricarboxylates, which is required in several metabolic processes such as primary amino acid synthesis (e.g. nitrate/ammonium assimilation), export of reducing equivalents (e.g. for photorespiration), fatty acid metabolism (e.g. lipid mobilization and fatty acid elongation), gluconeogenesis, and isoprenoid biosynthesis (5-14). However, protein (gene) sequences are known for only a few plant mitochondrial carriers. Indeed, cDNAs encoding the adenine nucleotide carrier, the uncoupling protein, and the phosphate carrier have been isolated from various plants based on the high homology with their orthologs in other organisms (15-17). The latter two carriers have been identified also from their transport properties upon expression in Escherichia coli and reconstitution into liposomes (18, 19). Finally, a malate translocator from Panicum miliaceum has been overexpressed, reconstituted, and inferred to be the plant ortholog of the bovine oxoglutarate/malate carrier, although the identity between them is low (32%) (20, 21).

In this paper, the identification and characterization of the plant mitochondrial dicarboxylate-tricarboxylate carrier (DTC)¹ is described. It is based on the identification of a cDNA from Arabidopsis thaliana and four cDNAs from Nicotiana tabacum encoding proteins related to the bovine oxoglutarate-malate carrier (22). These proteins have the characteristic features of the mitochondrial carrier family and are 83-84% identical in their sequences. They were overexpressed in bacteria, purified, and reconstituted into phospholipid vesicles, where they transport dicarboxylates (such as oxoglutarate, oxaloacetate, malate, and succinate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate) by a counter exchange mechanism. From their transport properties and phylogenetic analysis, it is concluded that these proteins are isoforms/orthologs of a novel mitochondrial transporter named DTC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Material and Growth Conditions-- A. thaliana (ecotype Columbia), N. tabacum cv. xanthi, Nicotiana tomentosiformis, and Nicotiana sylvestris were grown in a greenhouse under long day conditions (16 h of light/8 h of dark). Natural light was supplemented with white fluorescent light to provide 200 µmol of photons s¹ m². For nitrogen supply experiments, N. tabacum were grown aeroponically as described in Ref. 23.

cDNA Library Screening-- A N. tabacum cDNA library was screened as described in Ref. 24 using the SacII/AccI fragment of AtDTC as probe. Hybridization and washing conditions were as described in Ref. 23. Standard DNA manipulations were as described in Ref. 25.

Southern and Northern Blot Analyses-- Genomic DNA was prepared from leaves according to Ref. 26. Total RNA was isolated from various tissues of A. thaliana and N. tabacum using TRIzol (Invitrogen). For nitrogen supply experiments, total RNA was extracted from N. tabacum tissues as described in Ref. 23. Southern and Northern blot analyses were carried out as described in Ref. 24. RNA loading was checked either with ethidium bromide or a 2.7-kbp PstI/HindIII fragment of constitutive wheat 18 S from plasmid pHC79 (27). All of the radiolabeled probes were generated by random priming with the T7 Quick-prime^TM kit (Amersham Biosciences) and [³²P]dCTP (Amersham Biosciences). The membranes were autoradiographed at 70 °C for several days with an intensifying screen.

Bacterial Expression and Purification of DTC Proteins-- The coding sequences of NtDTC1, NtDTC2, NtDTC3, and AtDTC were amplified from the respective cDNAs by PCR. Forward and reverse oligonucleotide primers were synthesized corresponding to the extremities of the DTC coding sequences with additional NdeI and EcoRI sites, respectively. PCR products were cloned as NdeI/EcoRI fragments into the pMW7 expression vector. The resulting constructs were used to transform E. coli C0214(DE3) cells, and recombinant DTC proteins were overproduced as inclusion bodies at 37 °C as described before (28). Inclusion bodies were purified on a sucrose density gradient (22), washed at 4 °C with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 6.5), then washed twice with a buffer containing Triton X-114 (3%, w/v), 1 mM EDTA and 10 mM PIPES-NaOH, pH 6.5, and washed once again with TE buffer. DTC proteins were solubilized in 2.5% (w/v) sarkosyl, 0.1 mM EDTA, and 10 mM Tris-HCl, pH 7.0, and a small insoluble residue was removed by centrifugation at 258,000 × 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 (29). 1.43 mg/ml of soybean asolectin (Fluka) was included in the reconstitution mixture for optimal transport activity. External substrate was removed from proteoliposomes on Sephadex G-75 columns. Transport at 25 °C was started by adding [¹⁴C]oxoglutarate (PerkinElmer Life Sciences), [¹⁴C]malate, or [¹⁴C]citrate (Amersham Biosciences) to the proteoliposomes and terminated after 20 s by the addition of 30 mM pyridoxal 5'-phosphate and 10 mM bathophenanthroline (the "inhibitor-stop" method) (29). In controls, the inhibitors were added with the labeled substrate. The transport measurements were carried out at the same internal and external pH values of 6.0 (except where otherwise indicated). External radioactivity was removed from quenched samples on Sephadex G-75, and the internal radioactivity was measured (29). The transport activity was the difference between experimental and control values. K⁺ diffusion potentials were generated using valinomycin and K⁺ gradients. In these experiments the reconstitution mixture contained 0.71 mg/ml of cardiolipin (Sigma) besides soybean asolectin.

Other Methods-- Recombinant NtDTC2 protein with an MHHHHHHTM N-terminal extension was produced in E. coli BL21(DE3) cells, purified as described in Ref. 23, and used to raise polyclonal antibodies in rabbits (Eurogentec, Belgium). Submitochondrial fractions of potato tubers were prepared as described in (30). The proteins were analyzed by SDS-PAGE and stained with Coomassie Blue dye. The yield of the purified recombinant proteins per liter of bacterial culture was estimated by laser densitometry (28). The amount of protein incorporated into liposomes was measured as described (28). With all DTCs the efficiency of reconstitution (i.e. the share of successfully incorporated protein) varied between 18 and 23% of the protein added to the reconstitution mixture in all experiments. Protein transfer onto nitrocellulose membranes and immunodetection were performed as described in Ref. 24.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Characterization of DTC cDNAs-- The protein sequence of the bovine 2-oxoglutarate/malate transporter (22) was used to search data bases for homologous plant sequences. In this way an A. thaliana EST (GenBank^TM accession number Z26469), named AtDTC, was identified. Sequencing showed that it contained a 897-bp open reading frame, encoding a polypeptide of 298 amino acids with a calculated molecular mass of 31.9 kDa. AtDTC was used to screen a N. tabacum cDNA library, and four different cDNAs were identified (named NtDTC1 to NtDTC4). They were classed into two groups containing NtDTC1 with NtDTC2 and NtDTC3 with NtDTC4. Analysis of NtDTC1 showed an 894-bp open reading frame that encoded a 297-amino acid polypeptide with a calculated molecular mass of 31.7 kDa. The NtDTC2 cDNA was not complete, lacking six nucleotides after the start codon as well as a 5'-untranslated region. There was 99% amino acid identity between NtDTC1 and NtDTC2. In the second group, NtDTC3 gave a 903-bp open reading frame that encoded a 300-amino acid polypeptide with a calculated molecular mass of 32.1 kDa. NtDTC4 was not a complete cDNA, containing only a 682-bp coding region. There was 98% amino acid identity between NtDTC3 and NtDTC4. When the two groups of tobacco DTCs were compared, an amino acid identity of 91% was found. NtDTC1/2 and NtDTC3 exhibited 84 and 83% identity with AtDTC, respectively. Both AtDTC and the NtDTCs exhibit the hydrophobic profile, the tripartite structure, and the sequence motifs that are characteristic of the mitochondrial carrier family (1).

Number of Genes Coding for the DTC in A. thaliana and N. tabacum-- An analysis of the Arabidopsis nucleotide sequence data base showed the presence of a single AtDTC gene located on chromosome V (GenBank^TM accession number AF296838). The restriction enzymes EcoRI, BamHI, and HindIII, which do not have recognition sites within this gene, were used to digest A. thaliana genomic DNA. These digests produced a single fragment that hybridized with the AtDTC probe (Fig. 1A), thus confirming that the Arabidopsis genome contains a single copy of the AtDTC gene. The four NtDTCs were isolated from N. tabacum, an amphidiploid species derived from ancestors that are closely related to the present-day species N. sylvestris and N. tomentosiformis. Therefore, Southern analyses were performed using genomic DNA from N. tabacum, N. sylvestris, and N. tomentosiformis. A number of hybridizing bands were detected in the different tobacco genomes when using a nonspecific NtDTC probe, thus confirming that several distinct genes encode DTC in tobacco (Fig. 1B). Indeed, it appeared that N. sylvestris and N. tomentosiformis contained at least two DTC genes, whereas the N. tabacum pattern was the sum of these two genomes. Southern blotting with a specific NtDTC4 probe and PCR analyses carried out on genomic DNA using gene-specific oligonucleotide primers revealed that NtDTC2/3 and NtDTC1/4 were encoded by the N. sylvestris and N. tomentosiformis genomes, respectively (data not shown).

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Southern blot analysis of genomic DNA. A, DNA (15 µg) from A. thaliana was digested with EcoRI (E), HindIII (H), and BamHI (B) and hybridized with the SacII/AccI fragment of AtDTC. B, DNA (30 µg) from N. tabacum (N.tab), N. sylvestris (N.syl), and N. tomentosiformis (N.tom) were digested with SacI (S), EcoRI (E), and HindIII (H) and hybridized with the EcoRI/SacI fragment of NtDTC2. The molecular marker sizes (in kbp) are marked on the left side of each panel.

Molecular and Phylogenetic Relationship of AtDTC and NtDTC Proteins with Other Mitochondrial Transporters-- Amino acid sequence comparisons revealed that AtDTC and the NtDTCs showed the highest degree of similarity with other plant proteins. For example, NtDTC1 exhibited 94% identity with a putative potato oxoglutarate-malate carrier (GenBank^TM accession number X99853) and 81% identity with the millet malate transporter (20). Among mitochondrial transporters from other organisms, the tobacco DTCs showed the highest homology with the mitochondrial oxoglutarate-malate carriers sharing 41-42% identical amino acids with this carrier from bovine (22), human (31), rat (32), and Caenorhabditis elegans (GenBank^TM accession number P90992). A phylogenetic analysis carried out using AtDTC, NtDTC1-4, and the sequences of four groups of mitochondrial transporters (i.e. the oxoglutarate-malate carrier (OGC), dicarboxylate (DIC), and tricarboxylate (CTP) carriers, and the uncoupling (UCP) proteins) revealed that AtDTC and the NtDTCs were more closely related to the OGC group (Fig. 2). However, DTCs and OGCs form two clearly distinct clusters. Furthermore, the phylogenetic analysis confirmed the presence of two distinct DTC subgroups in tobacco (made up of the homologous DTC proteins from each of the two N. tabacum genomes) that appear to have originated from a recent gene duplication event.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Phylogenic tree of amino acid sequences of mitochondrial transporters from various organisms. The dendogram was constructed with Clustal X using the neighbor-joining method based on the sequence information of this work and mitochondrial carrier sequences retrieved from the GenBankTM and EMBL data bases. The proteins had the following accession numbers: yeast SFC, Z49595; C. elegans CTP, P34519; bovine CTP, P79110; human CTP, BC004980; rat CTP, P32089; yeast CTP, U17503; yeast DIC, U79459; C. elegans DIC, X76114; human DIC, AJ131613; rat DIC, AJ223355; mouse DIC, AF188712; putative potato oxoglutarate-malate carrier, X99853; millet malate carrier, D45073; putative C. elegans oxoglutarate-malate carrier, P90992; rat OGC, U84727; human OGC, X66114; bovine OGC, M60662; human UCP4, AF110532; potato UCP, Y11220; Arabidopsis UCP, AJ223983; wheat UCP, AB042429; mouse UCP3, AF032902; dog UCP3, AB022020; human UCP3, U82818; human UCP2, AF096289.

Expression of DTC in Various Plant Tissues-- The expression of DTC in A. thaliana and N. tabacum was examined in different tissues by Northern blot analysis. The presence of DTC transcripts was found in all plant tissues examined, although at different levels (Fig. 3). In Arabidopsis, the flower bud showed the highest DTC transcript levels, whereas the root had the lowest (Fig. 3A). In N. tabacum DTC expression in flower bud and root appeared to be comparable; the highest transcript levels were detected in sepal, petal, male, and female tissues, whereas somewhat lower amounts were found in leaf and stem (Fig. 3B). Although we were unable to distinguish between the different tobacco DTCs with the probe used in Fig. 3, reverse transcription-PCR experiments using gene-specific primers indicated that each tobacco DTC gene was expressed in the different organs/tissues analyzed (data not shown).

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Expression analysis of DTC mRNA in various plant tissues. Northern blots were carried out with total RNA (20 µg) extracted form tissues isolated from A. thaliana (A) and N. tabacum (B) plants. Total RNA was hybridized with the SacII/AccI fragment of AtDTC for A. thaliana and with the EcoRI/SacI fragment of NtDTC2 for N. tabacum. A wheat 18 S probe (18s) was used to check RNA loading in each lane. Total RNA was isolated from leaf (L), root (R), stem (S), flower bud (Bu), flower (Fl), sepal (Se), petal (Pe), male tissue (MT), and female tissue (FT).

Effect of Nitrate Supply on NtDTC Steady-state mRNA Levels-- Organic acids produced in the mitochondria are required for ammonium assimilation via the glutamine synthetase/glutamate synthase pathway located in the plastids (33). Previous work has shown a coordinated expression of certain genes (NAD-dependent isocitric dehydrogenase, nitrate reductase, and glutamine synthetase) when nitrate is supplied to nitrogen-starved tobacco plants (23). It was therefore interesting to investigate whether DTC expression varied under the same conditions. The effect of nitrate supply to tobacco plants that were nitrogen-starved for 4 days was investigated in roots (Fig. 4A) and deveined leaves (Fig. 4B) by Northern blot analysis as described in Ref. 23. In both tissues, nitrate resupply led to an increase in NtDTC mRNA steady-state levels (Fig. 4).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Changes in steady-state transcript abundance in roots and leaves after nitrate realimentation to 4-day nitrogen-starved tobacco plants. Total mRNA (20 µg) was extracted from nitrogen-starved roots (A) and deveined leaves (B) at 0, 3, 8, 23, and 32 h after nitrate resupply and hybridized with the EcoRI/SacI fragment of NtDTC2. The ethidium bromide-stained gel shows RNA loading in each lane.

DTC Is a Mitochondrial Membrane Protein-- To study whether the DTC protein is located in the membranes of plant mitochondria, a His₆-tagged NtDTC2 protein was purified by affinity chromatography (Fig. 5, lane 1) and used to raise polyclonal antibodies in rabbits. The antibodies recognized the recombinant DTC protein as shown by Western blot analysis (Fig. 5, lane 5). DTC could not be detected in crude tobacco leaf extracts (Fig. 5, lanes 2 and 6). In contrast, a prominent immunoreactive band of about 32 kDa was observed using purified mitochondria from potato tubers. To examine the submitochondrial location of DTC, integral membrane proteins were separated from soluble and peripheral proteins of potato mitochondria by carbonate treatment (Fig. 5, lanes 3 and 4, respectively). The 32-kDa band was found only in the membrane fraction (Fig. 5, lanes 3 and 7). Therefore, DTC is an integral mitochondrial membrane protein. The antiserum cross-reacted with another protein of about 60 kDa. However, this protein is not related to DTC because it was still recognized when the immunoreaction was carried out in the presence of soluble recombinant NtDTC2 (not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Expression of recombinant tobacco DTC2 in E. coli and subcellular localization of plant DTC. The protein extracts were subjected to 10% SDS-PAGE and analyzed by Coomassie Blue staining (lanes 1-4) and Western blotting with antibodies raised against the NtDTC2-His6 protein (lanes 5-8). Lanes 1 and 5, purified recombinant NtDTC2-His6 protein; lanes 2 and 6, crude extract from tobacco leaves; lanes 3 and 7, membrane proteins from S. tuberosum mitochondria; lanes 4 and 8, soluble proteins from S. tuberosum mitochondria; lane M, molecular weight marker proteins.

Functional Characterization of the Recombinant DTC Proteins-- To study their functional properties, NtDTC1-3 and AtDTC proteins were expressed at high levels in E. coli C0214 (DE3). They accumulated as inclusion bodies and were purified by centrifugation and washing with a yield of 80-100 mg of purified protein/liter of bacterial culture. Neither protein was detected in bacteria harvested immediately before induction of expression or in control (with only vector) cells harvested after induction (not shown). The identity of the expressed proteins was confirmed by N-terminal sequencing and Western blotting.

Proteoliposomes reconstituted with the recombinant DTC proteins from tobacco and A. thaliana catalyzed an active counterexchange of external [¹⁴C]oxoglutarate for internal oxoglutarate (Table I). No activity was detected with DTC proteins that had been boiled before incorporation into liposomes. Similarly, no oxoglutarate/oxoglutarate exchange was detected by reconstitution of sarkosyl-solubilized material from bacterial cells either lacking the DTC expression vector or harvested immediately before induction of expression. Furthermore, no uptake was observed of external [¹⁴C]oxoglutarate into liposomes that did not contain internal oxoglutarate, indicating that reconstituted DTCs do not catalyze unidirectional transport (uniport).

The substrate specificity of the tobacco and A. thaliana reconstituted proteins was investigated in detail by measuring the uptake of [¹⁴C]oxoglutarate into proteoliposomes that had been preloaded with potential substrates. As shown in Table I, the highest activities were detected in the presence of internal 2-oxoglutarate, malate, maleate, oxaloacetate, succinate, or malonate. Interestingly, citrate, isocitrate, cis-aconitate, trans-aconitate, and sulfate were exchanged for external 2-oxoglutarate, although to a slightly lower extent than the dicarboxylates. No significant exchange was detected with internal fumarate, phosphoenolpyruvate, phosphate, pyruvate, glutamate (Table I), aspartate, glutamine, carnitine, ornithine, or ADP (not shown). Similar results were obtained with NtDTC2 (data not shown).

The ability of nonradioactive substrates (at a concentration of 4 mM) to inhibit the uptake of 0.2 mM [¹⁴C]oxoglutarate into proteoliposomes containing 20 mM oxoglutarate and reconstituted with the DTC proteins was also examined. The oxoglutarate/oxoglutarate exchange was inhibited by the external addition of 2-oxoglutarate, malate, maleate oxaloacetate, succinate, malonate, citrate, isocitrate, cis-aconitate, and trans-aconitate (70-95% inhibition) and, to a lesser extent (35-40%), by oxoadipate. Sulfate, a potential DTC substrate (Table I), had a very low inhibitory effect (<20%), indicating that the reconstituted DTC proteins have a very low affinity for sulfate, at least at the external surface of the proteoliposomes. Phosphoenolpyruvate, fumarate, phosphate, pyruvate, glutamate, ornithine, carnitine, and ADP had a very limited inhibitory effect, if any. Therefore, the substrate specificity of the plant DTC proteins appears to be broader than that of known oxoglutarate-malate carriers, because DTC accepts both dicarboxylates and tricarboxylates.

The [¹⁴C]oxoglutarate/oxoglutarate exchange in proteoliposomes reconstituted with the tobacco and A. thaliana DTC proteins was inhibited strongly (61-98%) by organic mercurials, such as mersalyl (0.1 mM) and p-chloromercuriphenylsulfonate (0.1 mM), and by bathophenanthroline (1 mM) (inhibitors of several mitochondrial carriers). The impermeable dicarboxylate butylmalonate (2 mM), an inhibitor of the dicarboxylate and oxoglutarate/malate carriers (34, 35), and especially phthalonate (2 mM), an inhibitor of oxoglutarate and oxaloacetate transport in mitochondria (11, 13), inhibited (60-99%) the reconstituted transport activity. Furthermore, the tricarboxylate analog 1,2,3-benzenetricarboxylate (2 mM), a specific inhibitor of the tricarboxylate carrier (36), had a considerable inhibitory effect (61-65%), as did pyridoxal 5'-phosphate (1 mM, 45-60% inhibition). In contrast, N-ethylmaleimide (1 mM), -cyanocinnamate (0.1 mM), and carboxylatractyloside (0.02 mM), known inhibitors of the phosphate, pyruvate, and ADP/ATP carriers, respectively, had no effect on the activities of the reconstituted DTC proteins.

Effect of pH on DTC-catalyzed Transport-- The DTC-mediated oxoglutarate and citrate homoexchanges were found to be strongly dependent on pH (Fig. 6). With both NtDTC1 and AtDTC (Fig. 6), the oxoglutarate/oxoglutarate and citrate/citrate exchanges increased on decreasing the pH from 8.0 to 5.5. Below this pH value transport was inhibited, possibly because of carrier inactivation. Three additional points are noteworthy. First, oxoglutarate transport catalyzed by AtDTC (Fig. 6B) increased slightly on decreasing the pH from 7.0 to 5.5 as compared with oxoglutarate transport catalyzed by NtDTC1 (Fig. 6A). Second, citrate transport was very low above pH 7.0 with both reconstituted NtDTC1 and AtDTC (Fig. 6). Third, reconstituted NtDTC2 gave virtually the same results as those for NtDTC1 (Fig. 6A), whereas reconstituted NtDTC3 gave results qualitatively similar to those for AtDTC (Fig. 6B), although NtDTC3 transport rates were lower (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of pH on reconstituted DTC activity. Liposomes were reconstituted with recombinant NtDTC1 (A) or AtDTC (B). Transport was started by the external addition of 0.1 mM [14C]oxoglutarate () or 0.1 mM [14C]citrate () to proteoliposomes containing 20 mM oxoglutarate or citrate, respectively, and terminated after 20 s. Similar results were obtained in three independent experiments for each carrier.

Kinetic Characteristics of the Recombinant DTC Proteins-- The kinetic constants of reconstituted DTC from tobacco and Arabidopsis were determined at pH 6 and 7 by measuring the initial transport rate at various external [¹⁴C]oxoglutarate, [¹⁴C]malate, or [¹⁴C]citrate concentrations in the presence of a constant saturating internal concentration of the same substrate (homoexchange). The mean K_m and V_max values and their standard errors are shown in Table II. For all DTC proteins tested at pH 6, the maximum rates (V_max) of malate transport were higher than those of oxoglutarate and citrate, whereas oxoglutarate transport was intermediate. The apparent transport affinities (K_m) for citrate and oxoglutarate were virtually the same, whereas those for malate were higher (about 5-fold with NtDTC1 and about 2.5-fold with AtDTC). At pH 7, the K_m for oxoglutarate and malate as well as the V_max of oxoglutarate, malate, and citrate transport were only slightly different from those measured at pH 6. On the other hand, the K_m values for citrate were about 6-fold higher at pH 7 than at pH 6. 

Citrate and malate were found to be competitive inhibitors of NtDTC1-mediated oxoglutarate transport because they increased the apparent K_m without changing the V_max of [¹⁴C]oxoglutarate/oxoglutarate exchange. Similarly, oxoglutarate and malate were found to act as competitive inhibitors of the NtDTC1-catalyzed [¹⁴C]citrate/citrate exchange. The inhibition constants (K_i) at pH 6 were 0.30 ± 0.11 mM for oxoglutarate, 1.21 ± 0.36 mM for malate, and 0.29 ± 0.06 mM for citrate (means ± S.E. of eight experiments). In three other experiments, citrate and malate were found to inhibit the AtDTC-mediated oxoglutarate homoexchange in a competitive manner.

Substrate Species Transported by the DTC Proteins-- Citrate has pK_a values of 3.14, 4.77, and 5.40; between pH 6 and 7 citrate³ is the predominant species. As shown in Table II, in the pH range between 6 and 7, the V_max of citrate transport varied only slightly, whereas the apparent transport affinity decreased considerably with increasing pH. In principle, this pH dependence of citrate transport may be caused by a pH effect on the protein and/or by the availability of the transported substrate species. To investigate these possibilities, the K_m values for the different citrate species were calculated. Table III reports the data obtained for the reconstituted NtDTC1. It is clear that the K_m values for H-citrate² at pH 6 and 7 were similar (as is the maximal rate of citrate transport). In contrast, the K_m values for citrate³, H₂-citrate, and H₃-citrate are very different. Furthermore, the calculated K_m values for uncharged citrate were extremely low. These results indicate that H-citrate² is the species transported by NtDTC1. A similar conclusion was reached from the apparent transport affinities of NtDTC3 and AtDTC for the different citrate species calculated at pH 6 and 7 (data not shown). The effect of pH on the transport affinities of NtDTC1 for the different malate species was also examined. Malate has pK_a values of 3.36 and 5.10, and consequently malate² is the predominant species in the pH 6-7 range. The calculated K_m for malate² did not significantly change with pH; in contrast, the K_m value for H-malate and H₂-malate varied considerably (Table III). Again, the maximal rate of malate transport was only slightly affected. As in the case of citrate, these results argue for malate² as the transported species. The same argument has been used previously to show that H-citrate² and malate² are the actual substrates of the mitochondrial tricarboxylate carrier (37) and that H-citrate² is the actual substrate of the Klebsiella pneumoniae citrate transporter (38).

If H-citrate² and malate² (or oxoglutarate²) are the species transported by DTCs, their exchange should be electroneutral. Therefore, the influence of the membrane potential on the citrate/oxoglutarate heteroexchange was investigated. The membrane potential was generated across the proteoliposomal membrane as a K⁺ diffusion potential using valinomycin. The rates of [¹⁴C]citrate_out/oxoglutarate_in and [¹⁴C]oxoglutarate_out/citrate_in exchanges catalyzed by NtDTC1 were not influenced by the addition of valinomycin, neither in the absence nor in the presence of a K⁺ gradient of 1:50 (mM/mM, in/out) corresponding to a membrane potential of 100 mV positive inside (data not shown). Similar results were found with NtDTC2, NtDTC3, and AtDTC. In contrast, the [¹⁴C]aspartate_out/glutamate_in exchange mediated by the aspartate-glutamate carrier, which is known to catalyze an electrophoretic exchange between aspartate and glutamate + H⁺ (28), was stimulated about 3-fold by the addition of valinomycin in the presence of a K⁺ gradient 1:50 (mM/mM, in/out). Virtually no effect was observed in the absence of a potential (without K⁺ gradient) or when the aspartate/aspartate homoexchange was measured (data not shown). These observations indicate that DTC catalyzes an electroneutral exchange of oxoglutarate for citrate independently of the type of substrate present at each side of the membrane.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this work overexpression in E. coli of hitherto unidentified mitochondrial carriers and reconstitution of recombinant proteins in liposomes have been employed to study the transport properties and kinetic parameters of plant DTCs. The results demonstrate that the Arabidopsis and tobacco DTC proteins are isoforms/orthologs of a novel mitochondrial carrier that is capable of transporting both dicarboxylates (such as oxoglutarate, oxaloacetate, malate, succinate, maleate, and malonate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate). The specificity of DTC for dicarboxylates and tricarboxylates is demonstrated by the dependence of [¹⁴C]oxoglutarate transport on countersubstrates and by the sensitivity of this transport to externally added substrates and to impermeable analogs of dicarboxylates and tricarboxylates. DTC appears to share a common substrate-binding site for oxoglutarate, malate, and citrate, because these substrates are mutually competitive inhibitors and because their K_i values are close to the respective K_m values. Furthermore, DTC accepts only the single protonated form of citrate (H-citrate²) and the unprotonated form of malate (malate²) and catalyzes electroneutral exchanges, for example of a dicarboxylate for a tricarboxylate + H⁺. The more pronounced pH dependence from 7.0 to 5.5 of oxoglutarate transport catalyzed by NtDTC1 and NtDTC2, as compared with that of NtDTC3 and AtDTC, may be explained by a pH effect on one or more nonconserved residues between the DTC isoforms/orthologs, possibly histidine 145 of NtDTC1 and NtDTC2.

The substrate specificity of DTC is distinct from that of any other mitochondrial carrier of known sequence and function, including those for either dicarboxylates or tricarboxylates, although with these there is some degree of substrate overlap. The substrate specificity of DTC differs from that of its closest sequence homolog, the oxoglutarate/malate carrier (22), which does not transport citrate, isocitrate, cis-aconitate, trans-aconitate, and sulfate and transports oxaloacetate only very inefficiently. DTC differs from dicarboxylate (7, 39), succinate/fumarate (40), oxaloacetate (41), and oxodicarboxylate (42) carriers (the principal substrates of which are malate and phosphate, succinate and fumarate, oxaloacetate and sulfate, and oxoadipate and oxoglutarate, respectively) because DTC transports tricarboxylates efficiently, transports oxoadipate to a low extent, and does not transport phosphate and fumarate. It also differs from the tricarboxylate carrier (43, 44) because the latter transports only tricarboxylates (but not trans-aconitate), dicarboxylates (but not oxoglutarate and oxaloacetate), and phosphoenolpyruvate. Furthermore the DTC proteins share only 15 and 21% amino acid identity with the yeast (43) and the rat (44) tricarboxylate carriers, respectively.

The higher relatedness of DTC with oxoglutarate-malate carriers (41-42% identity), as compared with any other characterized carrier (Fig. 2), suggests that these transporters originated from a common ancestor. This ancestor duplicated in some organisms, for example in animals, giving rise to the oxoglutarate-malate carrier and the tricarboxylate carrier, whereas in plants it evolved into DTC, which exhibits the combined characteristics of both the oxoglutarate-malate and the tricarboxylate carriers. Several available plant protein sequences show a 81-94% identify with our DTC proteins, including the millet malate translocator (20) and a putative potato oxoglutarate/malate carrier (GenBank^TM accession number X99853) as well as many partial sequences, including AF010583 from Oryza sativa, BG444303 from Gossypium arboreum, BG645594 from Medicago truncatula, BG320815 from Zea mays, BE421905 from Hordeum vulgare, BF480205 from Mesembryanthemum crystallinum, BM437521 from Vitis vinifera, BF011089 from Beta vulgaris, BG526291 from Stevia rebaudiana, and BM411548 from Lycopersicon esculentum. In view of the high degree of identity between these proteins and DTC, we conclude that they are all dicarboxylate-tricarboxylate carriers.

It is interesting to note that the transport characteristics of the recombinant DTC from Arabidopsis and tobacco (two C3-type plants) closely resemble those of a 31-kDa mitochondrial protein purified from maize (a C4-type plant) (14). Indeed, this protein, when reconstituted into liposomes, transports citrate, isocitrate, cis-aconitate, oxoglutarate, oxaloacetate, malate, succinate, and malonate by an exchange mechanism. Furthermore, oxoglutarate and malate are competitive inhibitors with respect to citrate. Their inhibition constants (0.5 mM for oxoglutarate and 1.2 mM for malate) are very close to the K_i values determined in this work with the recombinant DTC. In the light of our findings it is likely that the purified 31-kDa protein from maize represents the DTC as both catalyze an active exchange of dicarboxylates and tricarboxylates. This hypothesis is supported by the observation that antibodies against the recombinant NtDTC2 cross-react with the purified maize protein (not shown), but more conclusive evidence is lacking because the N terminus of the protein is blocked, and insufficient amounts are available for mass spectrometry of digested peptides. In addition, proteoliposomes reconstituted with mitochondrial extracts from potato tuber were shown to catalyze a phthalonate-sensitive exchange of oxaloacetate for dicarboxylates (oxoglutarate, malate, and succinate) as well as for citrate (13). It was concluded that potato mitochondria contain an oxaloacetate translocator different from all other known mitochondrial transporters. We suggest that the above-mentioned transport activity is explained by DTC, which is present in potato (Fig. 5) and is largely spread in the plant kingdom (see above).

Because DTC transports a broad spectrum of dicarboxylates and tricarboxylates, this carrier may play a role in a number of important plant metabolic functions that require organic acid flux to or from the mitochondria. For example, the citrate exported from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. The malate/oxaloacetate exchange catalyzed by DTC can enable the export of redox equivalents from the mitochondrial matrix that can act as reductants for the production of glycerate during photorespiration in photosynthesizing cells. In agreement with a central role in cell metabolism, the expression pattern of DTC transcripts (Fig. 4) shows that it is present in all of the plant tissues/organs analyzed in this work. On the other hand, in P. miliaceum, a NAD-malic enzyme-type C₄ plant, the DTC ("malate transporter") gene was found to be specifically expressed in the bundle sheath cells of leaves where its expression increased with light and during greening in a manner similar to that of photosynthetic genes (44). These observations are in agreement with the involvement of DTC in the C4 pathway of photosynthesis. A further significance for DTC is the very likely possibility that it is involved in nitrogen assimilation, because 2-oxoglutarate is required for the assimilation of ammonium into amino acids by the glutamine synthetase/glutamate synthase pathway (33). To date, the exact enzymatic origin of this key organic acid for plant ammonium assimilation is still unknown (33). Two pathways have been proposed for the production of 2-oxoglutarate for ammonium assimilation, both requiring the export of organic acids from the mitochondria. In the first case, citrate is used to produce oxoglutarate in the cytosol by the concerted action of the cytosolic aconitase and NADP-dependent isocitrate dehydrogenase. In the second case, oxoglutarate is produced in the mitochondria by the NAD-isocitrate dehydrogenase. The observed increase in steady-state NtDTC transcript levels by the addition of nitrate to nitrogen-starved tobacco plants (Fig. 4) suggests a role of DTC in nitrogen assimilation. Under the same experimental conditions Lancien et al. (23) previously found an induction in the expression of NAD-isocitrate dehydrogenase, as well as citrate synthase, aconitase, nitrate reductase, and glutamine synthetase. The coordinated response of DTC and NAD-isocitrate dehydrogenase expression indicates that DTC is directly involved in the export of oxoglutarate from the mitochondria for nitrate assimilation.

	ACKNOWLEDGEMENT

We thank C. Colas des Francs for assistance in the preparation of the mitochondrial extracts.

	FOOTNOTES

^* This work was supported by the Consiglio Nazionale delle Ricerche (CNR) target project on Biotechnolgy, the European Social Fund, and grants from the Minstero dell'Istruzione, dell'Universita e della Ricerca (MIUR), MIUR-CNR L.95/96, and Centro di Eccellenza di Genomica comparata, University of Bari (CEGBA).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ311776, AJ311777, AJ311778, AJ311779, and AJ311780.

^§ These authors contributed equally to this work.

^¶ Supported by a Ministere d'Education National, de la Recherche et de la Technologie (MENRT) fellowship.

^** To whom correspondence should be addressed. Tel.: 39-080-5443323; Fax: 39-080-5442770; E-mail: fpalm@farmbiol.uniba.it.

Published, JBC Papers in Press, April 26, 2002, DOI 10.1074/jbc.M202702200

	ABBREVIATIONS

The abbreviations used are: DTC, dicarboxylate-tricarboxylate carrier protein; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); OGC, oxoglutarate-malate carrier; DIC, dicarboxylate carrier; CTP, tricarboxylate carrier; SFC, succinate-fumarate carrier; UCP, uncoupling protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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