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J. Biol. Chem., Vol. 277, Issue 22, 19289-19294, May 31, 2002

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Identification of the Mitochondrial Glutamate Transporter

BACTERIAL EXPRESSION, RECONSTITUTION, FUNCTIONAL CHARACTERIZATION, AND TISSUE DISTRIBUTION OF TWO HUMAN ISOFORMS^*

Giuseppe Fiermonte, Luigi Palmieri, Simona Todisco, Gennaro Agrimi, Ferdinando Palmieri^§, and John E. Walker^¶

From the  Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy and ^¶ The Medical Research Council-Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, United Kingdom

Received for publication, February 15, 2002, and in revised form, March 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H⁺ (or exchanged for OH), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H⁺ symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Functional studies in intact mitochondria have indicated that the inner membranes of mitochondria contain more than 20 transport proteins or carriers that catalyze the net flux or the exchange of physiologically important metabolites, nucleotides, and cofactors between the cytosol and the matrix (see Refs. 1 and 2 for reviews). In early studies, six mitochondrial carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, and the uncoupling protein were purified and sequenced (see Ref. 3 and references therein). Their sequences all consist of three tandemly repeated homologous domains, about 100 amino acids in length. All of the tandem repeats are related, and each of them contains a conserved sequence motif and two hydrophobic stretches that are thought to span the membrane as -helices. Therefore, they all belong to the same protein family (1, 4, 5). More recently, a large number of proteins of unknown function with the same characteristic features of this protein family have emerged from genome sequencing of various organisms. Since then, the functions of mitochondrial carriers for dicarboxylates, ornithine, succinate-fumarate, oxaloacetate-sulfate, oxodicarboxylates, deoxynucleotides, and aspartate-glutamate (6-13) and the adenine nucleotide transporter in peroxisomes of Saccharomyces cerevisiae have been identified by overexpression and reconstitution (14).

Other transport activities observed in whole mitochondria have yet to be associated with specific proteins. For example, the glutamate carrier (GC)¹ catalyzes the entry of glutamate into the mitochondrial matrix either with protons or in exchange for hydroxyl ions and plays important roles in amino acid degradation, nitrogen metabolism, and urea synthesis (see Refs. 15 and 16 for reviews). This activity is inhibited by bromocresol purple, tannic acid, N-ethylmaleimide, and other sulfhydryl reagents (17-20). The kinetics of glutamate transport have also been studied in mitochondria (19-22). The GC has a high K_m value and a low activity and was suggested to limit the effective activity of glutamate dehydrogenase, which is located exclusively in mitochondria, in the direction of glutamate deamination (19, 20, 23). The activity of glutamate transport was found to be higher in liver than in kidney (22) and heart (24), as anticipated from the central role of glutamate dehydrogenase in liver nitrogen metabolism.

Below, the identification of two isoforms, GC1 and GC2, of the mitochondrial GC is described. They are 323 and 315 amino acids long, respectively, have the characteristics of the mitochondrial carrier family, and are 63% identical in sequence.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequences Search and Analysis-- Eukaryotic data bases (www.ebi.ac.uk, ensembl.ebi.ac.uk, www.sanger.ac.uk, www.fruitfly.org, and www.ncbi.nlm.nih.gov) were screened with the sequences of the human isoforms of the aspartate/glutamate carrier (13) with BLASTP and TBLASTN. The amino acid sequences were aligned with ClustalW (version 1.7).

Construction of the Expression Plasmids-- The coding sequences for GC1 and GC2 were amplified by PCR from human brain cDNA (CLONTECH). The oligonucleotide primers were synthesized corresponding to the extremities of the coding sequences (accession numbers AK023106 (GC1) and AY008285 (GC2)) with additional NdeI and HindIII (GC1) and NdeI and EcoRI (GC2) sites. The amplified products were cloned into the pRUN expression vector (derived from pKN172 (25)), and the constructs were transformed into Escherichia coli TOP 10 cells (Invitrogen). Transformants were selected on 2xTY plates containing ampicillin (100 µg/ml) and screened by direct colony PCR and restriction digestion of plasmids. The sequences of inserts were verified.

Expression Analysis by Real-time PCR-- Total RNAs from human tissues (Invitrogen) were reverse-transcribed with the Gene Amp RNA PCR Core kit (Applied Biosystems) with random hexamers as primers. For real-time PCRs, primers and probes, based on the GC1 and GC2 cDNA sequences (accession number AK023106 and AY008285), were designed with Primer Express (Applied Biosystems). The forward and reverse primers for GC1 corresponded to nucleotides 1901-1923 and 1954-1969, respectively, and those for GC2 correspond to nucleotides 1650-1667 and 1715-1738, respectively. The GC1 FAM/MGB- and GC2 VIC/MGB-Dark Quencher-labeled probes corresponded to nucleotides 1925-1940 and 1679-1695 of the GC1 and GC2 cDNA sequences, respectively. Real-time PCRs were performed in a MicroAmp optical 96-well plate using the automated ABI Prism 7000 Sequence Detector System (Applied Biosystems). 50 µl of reaction volume contained: 5 µl of template (reverse-transcribed first-stranded cDNA), 1× TaqMan Universal Master Mix (Applied Biosystems), 200 nM probe for GC1 or GC2, respectively, and 900 nM of each primer. To correct for differences in the amount of starting first-stranded cDNAs, the human -actin gene (predeveloped assay reagents, Applied Biosystems) was amplified in parallel. The relative quantification of the two isoforms was performed according to the comparative method (2^Ct) (26),² with brain Ct as internal calibrator.

Bacterial Expression and Purification of GC1 and GC2-- GC1 and GC2 were obtained as inclusion bodies in E. coli CO214(DE3) (25, 27) and were purified as described previously (6, 13).

Transport Assays-- The recombinant proteins in sarkosyl were reconstituted into liposomes in the presence of 50 mM MES/50 mM HEPES at pH 6.0 (except where otherwise indicated) and with or without substrate (28). External substrate was removed from proteoliposomes on Sephadex G-75 columns, pre-equilibrated with 100 mM sucrose and 10 mM MES/10 mM HEPES at pH 6.0 (except where otherwise indicated). Transport at 25 °C was started by adding L-[U-¹⁴]glutamate (PerkinElmer Life Sciences) to substrate-loaded proteoliposomes (exchange) or to unloaded proteoliposomes (uniport). In both cases, transport was terminated by the addition of 30 mM pyridoxal 5'-phosphate and 10 mM bathophenanthroline (the "inhibitor-stop" method (28)). In controls, the inhibitors were added at the beginning together with the external substrate. Finally, the external substrate was removed, and the radioactivity in the liposomes was measured (28). The experimental values were corrected by subtracting control values. The initial transport rate was calculated from the radioactivity taken up by proteoliposomes after 3 min (in the initial linear range of substrate uptake). Alternatively, the initial transport rate was calculated from the time course of isotope equilibration (28). The reconstituted proteins were also assayed for other exchange activities (28).

Other Methods-- Proteins were analyzed by SDS-PAGE and stained with Coomassie Blue dye. N-terminal sequencing was carried out as described before (29). The amount of GC1 or GC2 was estimated by laser densitometry of stained samples using carbonic anhydrase as a protein standard (29). The amount of protein incorporated into liposomes was measured as described previously (29). About 18% of GC1 and GC2 was reconstituted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Glutamate Carrier Sequences-- The sequences of two isoforms of the human aspartate/glutamate carrier (13) were used to try and find candidates for the glutamate carrier. Several expressed sequence tags with p values between e⁵⁷ and e²⁰⁰ were discarded. They are probably orthologues of the aspartate/glutamate carrier. Two human expressed sequence tag sequences (AK023106 and AY008285) with p values of 3.2e⁴⁸ and 6.2e⁴⁶ encoded protein sequences of 323 (AK023106) and 315 (AY008285) amino acids. They were 63% identical to each other and 33% identical to the 393 amino acids of the C-terminal domains of the two isoforms of the aspartate/glutamate carrier that contain determinants of its transport activity and specificity (13). With these protein sequences, two conserved murine isoforms, AK010193 (323 amino acids) and Mm_101666 (315 amino acids), were identified that were 63% identical to each other. The protein sequence of AK010193 was 95 and 63% identical, respectively, to human AK023106 and AY008285, whereas the protein sequence of Mm_101666 was 63 and 87% identical to human AK023106 and AY008285, respectively. The related isoforms in the fruit fly (CG18347 and CG12201) and the nematode (F55G1.5 and F20D1.9) were less conserved, although in both, the isoform pair was 60-65% identical. Therefore, their correspondence with human and murine isoforms could not be deduced.

Expression of GC1 and GC2 in Tissues-- Real-time PCRs were performed on total RNA populations, using primers and probes from the cDNA sequences of GC1 and GC2 (Fig. 1). Both GC1 and GC2 were present at the same level in brain as they have the same threshold cycle value. This value was used as an internal calibration in the relative quantification of both isoforms. With the exception of brain, isoform 1 was expressed at higher levels than isoform 2. It was expressed most strongly in pancreas, liver, brain, and testis with lower levels in heart, kidney, lung, small intestine, and spleen. Isoform 2 was expressed in brain, to a lesser extent in testis, and poorly in all the other tissues. The ratio GC1:GC2 was 30 in liver, 28 in pancreas, 8.7 in spleen, 3.8 in kidney, 6.5 in small intestine, 2.5 in testis, 2 in heart, and 2.6 in lung.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Expression of human GC1 and GC2 in various tissues. Real-time PCR experiments were conducted on cDNAs prepared by the reverse transcription of total RNAs from various human tissues, using specific primers and probes based on human GC1 and GC2. The relative quantification of human GC1 (black bars) and GC2 (gray bars) was performed by the comparative method (2Ct). Human -actin was employed as reference.

Bacterial Expression of GC Proteins-- GC1 and GC2 were expressed at high levels in E. coli C0214(DE3) (Fig. 2). They accumulated as inclusion bodies and were purified by centrifugation and washing. The apparent molecular masses of the purified proteins (Fig. 2, lanes 3 and 6; yield 60-80 mg/l) were 32.8 kDa for GC1 and 31.5 kDa for GC2 (calculated values with initiator methionine, 34.5 and 33.8 kDa, respectively). Their identities were confirmed by N-terminal sequencing. The proteins were not detected in bacteria harvested immediately before the induction of expression (Fig. 2, lanes 1 and 4).

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Overexpression in E. coli and purification of human GC1 and GC2. Proteins were separated by SDS-PAGE and stained with Coomassie Blue. Lanes M, markers (ovotransferrin, bovine serum albumin, ovalbumin, carbonic anhydrase, myoglobine, and cytochrome c); lanes 1-2 and lanes 4-5, E. coli CO214(DE3) containing the expression vector with the coding sequence for GC1 (lanes 1 and 2) and GC2 (lanes 4 and 5). Samples were taken at the time of the induction (lanes 1 and 4) and 5 h later (lanes 2 and 5). Equivalent samples were analyzed. Lanes 3 and 6, 2 µg of GC1 and GC2 purified from bacteria in lanes 2 and 5, respectively.

Functional Characterization of GC1 and GC2-- The transport activities of GC1 and GC2 reconstituted in liposomes were tested in homo-exchange (same substrate inside and outside) experiments with substrates known to be transported by mitochondria. With external and internal substrate concentrations of 1 and 10 mM, respectively, both proteins catalyzed [¹⁴C]glutamate/glutamate exchange, inhibitable by a mixture of pyridoxal 5'-phosphate and bathophenanthroline. They did not catalyze homo-exchanges for phosphate, pyruvate, ADP, ATP, malonate, succinate, malate, oxoglutarate, ketoisocaproate, citrate, carnitine, aspartate, ornithine, lysine, arginine, histidine, glutathione, choline, spermine, proline, and threonine. No [¹⁴C]glutamate/glutamate exchange activity was observed with GC1 and GC2 that had been boiled before incorporation into liposomes nor by reconstitution of sarkosyl-solubilized material from bacterial cells either lacking the expression vector for GC1 or GC2 or harvested immediately before the induction of expression.

In Fig. 3, the kinetics are compared for the uptake by proteoliposomes of 1 mM [¹⁴C]glutamate measured either as uniport (in the absence of internal glutamate) or as exchange (in the presence of 10 mM glutamate). Both the exchange and the uniport reactions catalyzed by GC1 and GC2 followed first-order kinetics, isotopic equilibrium being approached exponentially. The ratio of maximal substrate uptake by the exchange and by the uniport was 8.3 for GC1 and 9.5 for GC2, in good agreement with the expected value of 10 from the intraliposomal concentrations at equilibrium (1 and 10 mM for uniport and exchange, respectively). The initial rates of glutamate uptake deduced from the time courses (28) were 20.1 and 1.4 µmol/min/g of protein for GC1 and 17.1 and 3.0 µmol/min/g of protein for GC2, for the exchange and the uniport reaction, respectively. The addition of 10 mM unlabeled glutamate after 90 min of incubation, when radioactive uptake by the proteoliposomes had approached equilibrium, caused an extensive efflux of radiolabeled glutamate from both glutamate-loaded and unloaded proteoliposomes (data not shown). This efflux shows that the [¹⁴C]glutamate taken up by uniport is released by exchange for externally added substrate. Therefore, the above reported results indicate that reconstituted GC1 and GC2 catalyze both the unidirectional transport of glutamate and the glutamate/glutamate exchange, as has been reported for the glutamate carrier studied in intact mitochondria (see Refs. 15 and 16 and references therein).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Kinetics of [14C]glutamate uniport and [14C]glutamate/glutamate exchange by GC1 and GC2. Proteoliposomes were reconstituted with GC1 (panel A) or GC2 (panel B). 1 mM [14C]glutamate was added to proteoliposomes containing 10 mM glutamate (exchange, ) or 10 mM NaCl and no substrate (uniport, ).

The substrate specificity of reconstituted GC1 and GC2 was examined in detail by measuring the uptake of [¹⁴C]glutamate into proteoliposomes that had been preloaded with various potential substrates (Table I). With both proteins, external L-glutamate exchanged significantly only in the presence of internal L-glutamate. A very low exchange (one order of magnitude lower than the glutamate/glutamate exchange) was found in the presence of internal -aminoadipate, adipate, glutarate, and -methylglutamate. Virtually no exchange was observed with the structurally related compounds L-aspartate, -aminopimelate, L-homocysteinesulfinate, L-cysteinesulfinate, L-glutamine, L-asparagine, D-glutamate, and D-aspartate (Table I) and with taurine, tartrate, cysteine, fumarate, malonate, succinate, L-malate, pimelate, phosphate, oxoglutarate, citrate, ATP, sulfate, oxaloacetate, pyruvate, phosphoenolpyruvate, L-carnitine, L-ornithine, or L-citrulline (not shown). The residual activity in the presence of these substrates was approximately the same as the activity observed in the presence of sodium chloride. Reconstituted GC1 and GC2, therefore, display a very narrow substrate specificity, which is confined virtually only to L-glutamate.

The [¹⁴C]glutamate/glutamate exchange reactions catalyzed by reconstituted GC1 and GC2 were inhibited strongly by pyridoxal 5'-phosphate, bathophenanthroline, mersalyl, and p-hydroxymercuribenzoate (inhibitors of several mitochondrial carriers) as well as by N-ethylmaleimide, tannic acid, and bromocresol purple, which are known inhibitors of the native GC (Fig. 4). In contrast, carboxylatractyloside, bongkrekate, 1,2,3-benzenetricarboxylate, and -cyano-4-hydroxycinnamate, inhibitors of other characterized mitochondrial carriers, had no or little effect on the activities of reconstituted GC1 or GC2.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of inhibitors on the [14C]glutamate/glutamate exchange by GC1 and GC2. Proteoliposomes were preloaded internally with 20 mM glutamate, and transport was initiated by adding 1 or 0.25 mM [14C]glutamate to proteoliposomes reconstituted with GC1 (filled bars) or GC2 (open bars), respectively. The incubation time was 3 min. The inhibitors were added 3 min before the labeled substrate. The final concentrations of the inhibitors were 50 µM (MER, mersalyl; p-HMB, p-hydroxymercuribenzoate), 10 mM (PLP, pyridoxal 5'-phosphate; BAT, bathophenanthroline), 1 mM (NEM, N-ethylmaleimide), 0.1% (TAN, tannic acid), 0.1 mM (BrCP, bromocresol purple), 2.5 mM (BTA, benzene-1,2,3-tricarboxylate), 20 µM (CCN, -cyanocinnamate), and 10 µM (CAT, carboxylatractyloside; BKA, bongkrekic acid). The extents of inhibition (%) from a representative experiment for each carrier are given. Similar results were obtained in at least three experiments.

Since the glutamate carrier catalyzes a glutamate + H⁺ co-transport across the mitochondrial membrane (see Refs. 15 and 16 for reviews), the influence of the proton gradient on the uptake of glutamate into unloaded liposomes reconstituted with GC1 or GC2 was investigated. In these experiments, the proton gradient across the proteoliposomal membrane was imposed as follows. The proteoliposomes were reconstituted at various pH values (from 6.0 to 8.0). After removal of the external buffer by passage through Sephadex G-75, [¹⁴C]glutamate (buffered at pH 6) was added to the proteoliposomes, and the initial rates of uptake were measured. The results, shown in Fig. 5, A and B, demonstrate that with both GC1 and GC2, the rate of glutamate uptake increased several times on increasing the internal pH from 6.0 to 8.0 (at a fixed external pH of 6.0). As a control, we investigated the dependence of the glutamate/glutamate exchange on the proton gradient imposed across the liposomal membrane using the same experimental conditions. Fig. 5, A and B, demonstrates that with both GC1 and GC2, the rate of glutamate/glutamate exchange was very little affected by the increase in the internal pH from 6.0 to 8.0 at a fixed external pH of 6.0. These experiments, showing that alkalinization of the compartment opposite to where glutamate is present stimulates markedly the net transport of glutamate but influences scarcely the glutamate/glutamate exchange, clearly indicate that glutamate is transported across the reconstituted liposomal membrane together with H⁺ or in exchange for OH. Thus, the recombinant GC1 and GC2 are also similar in these respects to the glutamate carrier described from studies in intact mitochondria (see Refs. 15 and 16 and references therein).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Dependence on trans-membrane pH gradient of the glutamate uniport by GC1 and GC2. The reconstitution mixture contained 20 mM glutamate (glutamate/glutamate exchange) or 10 mM NaCl (glutamate/H+ symport) and 50 mM MES/50 mM HEPES at the indicated pH values. After reconstitution of GC1 or GC2 into liposomes, a mixture of 100 mM sucrose and 0.1 mM MES/0.1 mM HEPES at the same pH of the reconstitution mixture was used to equilibrate and to elute the Sephadex G-75 columns. Transport was started by adding 1 or 0.5 mM [14C]glutamate together with 10 mM MES/10 mM HEPES at pH 6.0 to proteoliposomes reconstituted with GC1 (panel A) or with GC2 (panel B), respectively. The reaction was terminated after 3 min. , glutamate/H+ symport; , glutamate/glutamate exchange. Similar results were obtained in three independent experiments in duplicate for each carrier.

Kinetic Characteristics-- The kinetic constants of the recombinant purified GC1 and GC2 were determined by measuring the initial transport rate at various external [¹⁴C]glutamate concentrations in the presence of a constant saturating internal concentration of 20 mM glutamate (glutamate/glutamate exchange) or in the absence of internal substrate (glutamate/H⁺ symport) (Table II). When measured as glutamate/H⁺ symport at the same internal and external pH of 6.0, the transport affinities (K_m) of reconstituted GC1 and GC2 were 5.18 ± 0.48 and 0.26 ± 0.04 mM, respectively, and their specific activities (V_max) were 12.2 ± 2.9 and 3.9 ± 1.0 µmol/min/g of protein, respectively. Virtually the same K_m values were found for both GC1 and GC2 when the exchange mode of transport, instead of symport, was analyzed under the same experimental conditions. In contrast, with both isoforms, the V_max values were much higher for the glutamate/glutamate exchange transport mode (143.5 ± 32.8 µmol/min/g of protein for GC1 and 34.2 ± 6.5 µmol/min/g of protein for GC2), indicating that the glutamate/H⁺ symport is limited by the return of the unloaded carrier to the outward facing configuration (Table II). In mitochondria, the glutamate/glutamate exchange was also found to be much faster than the net uptake (16). In the presence of a trans-membrane pH gradient (external pH of 6 and internal pH of 8), the V_max values of the glutamate/H⁺ symport for both GC1 and GC2 were 4-5 times higher, whereas the corresponding K_m values for glutamate remained unchanged (Table II). In contrast, the V_max values of the glutamate/glutamate exchange were increased only 1.17-fold for GC1 and 1.23-fold for GC2 on increasing the internal pH from 6 to 8 at a fixed external pH of 6. Therefore, with both GC1 and GC2, the V_max value of the glutamate/H⁺ symport is markedly stimulated by the trans-membrane pH gradient, whereas the V_max value of the glutamate/glutamate exchange is almost unaffected, as observed for carriers that catalyze a proton symport.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The existence of a specific transporter for glutamate was inferred first in 1967 from studies of the reduction of mitochondrial NAD(P) by glutamate and of the swelling of liver mitochondria in ammonium glutamate (30). Since then, its main properties have been studied in intact mitochondria (15-24). However, the protein responsible for the glutamate carrier had not been identified hitherto. From the present study, it is clear that we have identified GC1 and GC2 as distinct isoforms of the glutamate carrier. They have molecular masses close to the usual 30 kDa of most mitochondrial carriers, and both have the tripartite structure and the sequence motif that are characteristic of the mitochondrial carrier family (1, 4, 5). The substrate specificity, inhibitor sensitivity, and pH dependence properties of reconstituted GC1 and GC2 match fully those of the native glutamate carrier. However, the two GC isoforms differ markedly in their kinetic parameters. GC1 has a very high K_m value for glutamate, whereas the K_m value of GC2 is low. The K_m value of GC1 (about 5 mM) is virtually identical to the K_m values for glutamate uptake (of 4-5 mM) measured in liver and kidney mitochondria (19-22). This K_m value of GC1 is much higher than those of other mitochondrial anion carriers (see Ref. 19 and references therein). It may be correlated to the high cytosolic concentration of glutamate, which in liver is of the same order of magnitude as the K_m value (31, 32). The low K_m value for glutamate was probably overlooked in the early studies with intact mitochondria because GC1 is more abundant than GC2 in all tissues (except brain) and particularly in liver (Fig. 1), and the V_max value of GC1 is higher than that of GC2 (Table II).

The almost exclusive substrate for both human GC isoforms is L-glutamate. Therefore, the main physiological role of GC isoforms is to import glutamate from the cytosol, where it is produced by the transamination of amino acids with oxoglutarate, to the mitochondrial matrix, where glutamate dehydrogenase is exclusively located. Although the V_max value of glutamate transport measured in intact mitochondria is lower than those of the other anion transporters (see Ref. 19 and references therein), it has been calculated that the maximal velocity of glutamate transport in rat liver mitochondria is high enough to feed the urea cycle, even if glutamate were the only ammonium source for the mitochondrial synthesis of carbamyl phosphate (19, 20). It should be emphasized that GC is the only mechanism for the supply of external glutamate to the intramitochondrial glutamate dehydrogenase since glutamate entering via the aspartate/glutamate exchanger is necessarily transaminated with intramitochondrial oxaloacetate to form aspartate. Since glutamate is co-transported with a proton by the GC and, therefore, its distribution across the mitochondrial membrane is influenced by pH, the entry of glutamate is favored in energized mitochondria. However, when ammonia rather than glutamate is the major nitrogen source for urea synthesis or when glutamine is generated intramitochondrially, for example by proline oxidation, the GC may operate in the reverse direction to limit the intramitochondrial accumulation of glutamate (21, 33). Furthermore, prevention of glutamate efflux from mitochondria by cytosolic H⁺ in kidney tubular cells may play an important role in the kidney response to metabolic acidosis by contributing to increased flux through glutamate dehydrogenase and hence to increased ammonia production (and excretion) from glutamine (16, 22).

The tissue specificity of the two GC isoforms differs markedly from that of the isoforms of the phosphate carrier (29), the ADP/ATP carrier (34-37), and other mitochondrial proteins involved in oxidative phosphorylation, such as the human -subunit of the ATP synthase complex (38), the mammalian proteolipid subunit of the ATP synthase (39), and the three mammalian subunits (VIa, VIIa, and VIII) of cytochrome oxidase (40, 41). The common feature of all these proteins is the presence of at least one heart type isoform, which is expressed abundantly only in muscles, and one liver type isoform, which is expressed ubiquitously in all tissues. The isoforms of GC are both ubiquitous, and GC1 is expressed in higher amounts than GC2 in all the tissues (except brain) and is particularly abundant in liver and pancreas. In the light of the observed differences in the kinetic parameters of the two isoforms, it appears that GC2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and GC1 becomes operative to accommodate the higher demands associated with specific metabolic functions, such as ureogenesis, and/or with special metabolic conditions, for example, after protein-rich diets. Thus, when the capacity of isoform GC2, which has a higher affinity for glutamate, is overwhelmed, isoform GC1 with its lower substrate affinity may be brought into operation by increased cytosolic glutamate concentration. Therefore, GC1 and GC2, with their different K_m and V_max values, can modulate the rate of glutamate transport into mitochondria and thus satisfy the tissue-specific metabolic demands.

	FOOTNOTES

^* This work was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, by MURST L.488/92 CO3 and CO4 and MURST-CNR L.95/95, by the Centro di Eccellenze di Genomica Comparate, University of Bari, by the Consiglio Nazionale delle Ricerche target project on Biotechnology, and by the European Social Fund.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.

This paper is dedicated to the memory of Professor Vincenzo Bocchini.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ428202, AJ428203

^§ To whom correspondence may be addressed. Tel.: 39-805443374; Fax: 39-805442770; E-mail: fpalm@farmbiol.uniba.it.

To whom correspondence may be addressed. Tel.: 44-1223252703; Fax: 44-1223252705; E-mail: walker@mrc-dunn.cam.ac.uk.

Published, JBC Papers in Press, March 15, 2002, DOI 10.1074/jbc.M201572200

² User Bulletin No.2 P/N 4303859, Applied Biosystems, Foster City, CA.

	ABBREVIATIONS

The abbreviations used are: GC, glutamate carrier protein; MES, 2-(N-morpholino)ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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