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Identification of the Human Mitochondrial Oxodicarboxylate Carrier

2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês

10.1074/jbc.m009607200

1083-351X

Giuseppe Fiermonte, Vincenza Dolce, Luigi Palmieri, Mario Ventura, Michael J. Runswick, Ferdinando Palmieri, John E. Walker,

Metabolomics and Mass Spectrometry Studies

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysineAJ278148AJ289714. In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysineAJ278148AJ289714. oxodicarboxylate carrier protein base pair(s) expressed sequence tag fluorescence in situ hybridization kilobase(s) nucleotide polyacrylamide gel electrophoresis reverse transcription polymerase chain reaction In mammals, 2-oxoadipate is produced from lysine in the cytosol of cells via the saccharopine and the pipecolic acid pathways. Catabolites of hydroxylysine and tryptophan enter these pathways as 2-aminoadipic-δ-semialdehyde and 2-oxoadipate, respectively. In the matrix of mitochondria, 2-oxoadipate is decarboxylated to glutaryl-CoA by the 2-oxoadipate dehydrogenase complex and then converted to acetyl-CoA. Over the years, several patients with 2-oxoadipate acidemia, an in-born error of metabolism of lysine, tryptophan, and hydroxylysine, have been reported, most of them slightly to deeply mentally retarded with hypotonia or seizures (1Przyrembel H. Bachmann D. Lombeck I. Becker K. Wendel U. Wadman S.K. Bremer H.J. Clin. Chim. Acta. 1975; 58: 257-269Crossref PubMed Scopus (55) Google Scholar, 2Wendel U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 3Wilson R.W. Wilson C.M. Gates S.C. Higgins J.V. Pediatr. Res. 1975; 9: 522-526Crossref PubMed Scopus (31) Google Scholar, 4Wilson R.W. Wilson C.M. Higgins J.V. Clin. Chim. Acta. 1976; 69: 323-332Crossref PubMed Scopus (9) Google Scholar, 5Fischer M.H. Brown R.R. Am. J. Med. Genet. 1980; 5: 35-41Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar, 7Vianey-Liaud C. Divry P. Cotte J. Teyssier G. J. Inherit. Metab. Dis. 1985; 8: 133-134Crossref PubMed Scopus (10) Google Scholar). It was speculated without experimental verification that their abnormal levels of serum and urinary 2-oxoadipate might arise from a defect in 2-oxoadipate dehydrogenase. As this enzyme is found in the matrix of mitochondria, and 2-oxoadipate is produced in the cytosol (8Nishizuka Y. Ichiyama A. Hayaishi O. Methods Enzymol. 1970; 17A: 463-491Crossref Scopus (32) Google Scholar), impaired transport of 2-oxoadipate into the organelle provides an alternative explanation, but until the present work no such transport function had been demonstrated in man. In this paper, the identification of the human 2-oxoadipate mitochondrial carrier (ODC)1is described. It is based on two isoforms, ODC1 and ODC2 encoded in the genome of Saccharomyces cerevisiae, that transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate across the inner membranes of mitochondria (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, yeast and man are too distant phylogenetically for these isoforms to provide a basis for direct cloning of the mammalian counterpart. Therefore, orthologs were sought and detected in Caenorhabditis elegans and Drosophila melanogaster and used to bridge between yeast and man. In this way, a human EST was identified that encodes a fragment of a related protein. It provided information to complete the human cDNA sequence. The encoded protein is 299 amino acids long and has the characteristic features of the family of mitochondrial carrier proteins (10Krämer R. Palmieri F. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers B. V., Amsterdam1992: 359-382Google Scholar, 11Walker J.E. Curr. Opin. Struct. Biol. 1992; 2: 519-526Crossref Scopus (105) Google Scholar, 12Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 13Palmieri F. van Ommen B. Papa S. Guerrieri F. Tager J.M. Frontiers of Cellular Bioenergetics. Kluwer Academic/Plenum Publishers, New York1999: 489-519Crossref Google Scholar). It was overexpressed in Escherichia coli, reconstituted into phospholipid vesicles, and shown to have transport specificity and other biochemical properties similar to those of the recombinant yeast ODC isoforms, including transport of 2-oxoadipate. Data bases of the genomes ofC. elegans and D. melanogaster at the Sanger Center (Hinxton, United Kingdom) and at the National Center for Biotechnology Information (Washington, D. C.) were screened with the sequences of the S. cerevisiae ODC proteins (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) using the program BLASTP. The NCBI nonredundant EST human data base was probed with the program TBLASTN. Amino acid sequences were aligned with ClustalW (version 1.7). Touchdown PCRs (14Don R.H. Cox P.T. Wainwright B.J. Baker K. Mattick J.S. Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2240) Google Scholar) were performed with adaptor-ligated double-stranded human liver cDNA (1 ng, CLONTECH), as described previously (15Indiveri C. Iacobazzi V. Giangregorio N. Palmieri F. Biochem. J. 1997; 321: 713-719Crossref PubMed Scopus (127) Google Scholar, 16Fiermonte G. Palmieri L. Dolce V. Lasorsa F.M. Palmieri F. Runswick M.J. Walker J.E. J. Biol. Chem. 1998; 273: 24754-24759Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The full-length cDNA sequence was obtained in two PCR reactions of 5′- and 3′-extension using the adaptor primers AP1 and AP2 (CLONTECH) and a primer set 1F, 2F, 1R, and 2R (Fig. 1) designed from the nucleotide sequence of the human EST, R29313. The PCR products were identified, recovered from agarose gels, cloned into the pCR2.1 topo-vector (Invitrogen), and sequenced. The cDNA for rat ODC was amplified similarly with oligonucleotides based on the human cDNA sequence. Human metaphase chromosome spreads were obtained from phytohemagglutinin-stimulated peripheral lymphocytes. A probe for FISH analysis was made by PCR amplification of human genomic DNA using primers corresponding to nt 916–939 (forward sense) and nt 1191–1212 (reverse sense) of the human cDNA for ODC (see Fig. 1). The resulting 4.2-kb fragment was cloned in the pCR2.1 topo-vector and sequenced to confirm its identity. The FISH experiments were performed as described previously (17Pannone E. Fiermonte G. Dolce V. Rocchi M. Palmieri F. Cytogenet. Cell Genet. 1998; 83: 238-239Crossref PubMed Google Scholar). Total RNAs (2 μg) were extracted and reverse-transcribed with the Gene Amp RNA PCR Core kit (PerkinElmer Life Sciences) using either random hexamers or oligo(dT)16 as primers (final volume, 40 μl). A 230-bp fragment of the ODC cDNA was then amplified from the reverse transcription reaction products (20 μl) by 35 cycles of PCR using oligonucleotides RT1F (nt 662–682) and RT1R (nt 869–892) as forward and reverse primers, respectively (Fig. 1). The products were probed with the radiolabeled oligonucleotide RT1P (nt 804–825; Fig. 1). As a control, a 384-bp β-actin fragment was amplified from the remainder of the reverse transcription products (18Dolce, V., Fiermonte, G., Runswick, M. J., Palmieri, F., and Walker, J. E. (2001) Proc. Natl. Acad. Sci. U. S. A., 98, in press.Google Scholar). The coding regions for the human and rat ODCs were amplified from human and rat liver cDNAs (1 ng) by 35 cycles of PCR. The forward and reverse primers in these reactions corresponded to nt 311–331 and 1188–1210 of the human ODC cDNA (see Fig. 1) and to nt 100–120 and 976–996 of the rat ODC cDNA (deposited as GenBankTMaccession number AJ289714). The forward and reverse primers carried anNdeI and a XhoI site, respectively, at their 5′-ends. The 0.9-kb products were gel-purified and cloned into the pRUN expression vector. Transformants of E. coli DH5α cells were selected on ampicillin (100 μg/ml) and screened by direct colony PCR and restriction digestion of plasmids. The sequences of inserts were verified. The overproduction of the ODC as inclusion bodies in the cytosol of E. coli was accomplished as described first for the bovine oxoglutarate-malate carrier (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar), except that the host cells were E. coli CO214(DE3) (20Palmieri L. De Marco V. Iacobazzi V. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 410: 447-451Crossref PubMed Scopus (80) Google Scholar). Control cultures with the empty vector were processed in parallel. Inclusion bodies were isolated, and ODC was purified by centrifugation and washing steps as described previously (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar, 21Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The recombinant protein in Sarkosyl was reconstituted into liposomes in the presence of substrates, as described previously (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). External substrate was removed from proteoliposomes on a Sephadex G-75 columns. The transport activity at 25 °C was determined by measuring the uptake (forward exchange) or the efflux (backward exchange) of [14C]oxoglutarate in exchange for unlabeled counter-substrates (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). For backward exchange measurements the proteoliposomes containing 1 mm internal oxoglutarate were prelabeled, after reconstitution, by carrier-mediated exchange equilibration by adding 10 μm[14C]oxoglutarate (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). After 30 min, the residual external radioactivity was removed by passing the proteoliposomes through a column of Sephadex G-75. In forward exchange reactions, transport was started by adding [14C]oxoglutarate to the proteoliposomes and in the backward exchanges by adding nonradioactive substrate. In both cases, transport was stopped after 1 min (in the initial linear range of substrate exchange) by addition of 10 mm pyridoxal 5′-phosphate and 10 mmbathophenanthroline (the "inibitor-stop" method (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar)). 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 (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). In forward exchange measurements, the experimental values were corrected by subtracting control values, and the rate was calculated in millimoles/min/gram of protein. In the case of backward exchanges, the rate in Δcpm/min was obtained from the decrease of internal radioactivity in 1 min. The reconstituted protein was assayed for other exchange activities by the inhibitor-stop method (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). Proteins were analyzed by SDS-PAGE and stained with Coomassie blue dye. N-terminal sequencing was carried out as described previously (21Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The amount of pure ODC was estimated by laser densitometry of stained samples (21Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The amount of ODC incorporated into liposomes was measured as described previously (23Fiermonte G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: 22782-22787Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and varied between 29 and 37% of the protein added to the reconstitution mixture. Western blotting was carried out as described previously (24Bisaccia F. Capobianco L. Brandolin G. Palmieri F. Biochemistry. 1996; 33: 3705-3713Crossref Scopus (38) Google Scholar) with a rabbit antiserum raised against the bacterially expressed rat ODC protein. By screening data bases, nematode and fruit fly clones (R11.1 and AAF45544.1) were found with 36 and 38% of the residues of their encoded proteins identical, respectively, to the yeast ODC protein sequences. A human EST data base was interrogated with these sequences, and a clone of 286 bp (R29313) was identified that encoded a protein sequence that was 62 and 68% identical, respectively, to regions of the C. elegans and the D. melanogaster proteins. The human EST was extended in the 5′- and 3′-directions by two PCR experiments. Among the products of the 5′-extension reaction, one band of about 1200 bp hybridized with primer 2F. Its sequence overlapped the EST. The 3′-extension product contained a single band of about 950 bp. Its sequence overlapped the 5′-extension. The final human cDNA sequence of 2024 nucleotides (Fig. 1) consisted of a 310-bp 5′-untranslated region, followed by an open reading frame of 900 bp, a 814-bp 3′-untranslated region containing a polyadenylation signal at nt 1974–1979 (25Proudfoot N.J. Brownlee G.G. Nature. 1976; 263: 211-214Crossref PubMed Scopus (1906) Google Scholar), and a poly(A)+ tail (see Fig. 1). The ATG codon at nt 311–313 is preceded by an in-frame stop codon 102-bp upstream and is likely to be the translational initiation codon. The N terminus of the human protein is at approximately the same position as in the rat, yeast, nematode, and D. melanogaster orthologs, confirming this view. The open reading frame encoded a polypeptide of 299 amino acids with a calculated isoelectric point of 9.6 and a molecular mass of 33,300 Da. The cDNA for the rat ODC was cloned in a similar way. This sequence (GenBankTM accession number AJ289714) consists of 1456 nucleotides with a 99-bp 5′-untranslated region, followed by an open reading frame of 897 bp, and a 460-bp 3′-untranslated region, containing neither a polyadenylation signal nor a poly(A)+tail. The open reading frame encoded a polypeptide of 298 amino acids with a molecular mass of 33,276. The human ODC is 82, 56, 55, 32, and 33% identical to the R. norvegicus, C. elegans,D. melanogaster, and S. cerevisiae ODC1 and ODC2 proteins, respectively. The gene for human ODC was found on chromosome 14 at 14q11.2 by FISH experiments conducted on normal human metaphase chromosomes (Fig.2). There was no evidence in this experiment for a second gene encoding an isoform, and no isoforms were detected in searches of data bases of human, nematode, and fruit fly sequences. The tissue distribution of mRNAs for the human and rat ODCs was studied by RT-PCR performed on total RNA populations, using primers and probes from regions of identity between the human and the rat nucleotide sequences. The ODC was detected in all tissues that were examined (Fig.3 A, panel a). The relatively weak signal from heart arises because less total RNA was employed than in other tissues, as the control demonstrates (seepanel b in Fig. 3 A). A similar pattern of expression was observed by Western blot analysis of rat mitochondria (see Fig. 3 B, panel a). The human ODC was overexpressed in E. coli C0214(DE3) (see Fig.4, lane 4) in the form of inclusion bodies. The purified protein gave a single band by SDS-PAGE (Fig. 4, lane 5) with an apparent molecular mass of 32 kDa. The protein was not detected in bacteria harvested immediately before induction of expression (Fig. 4, lanes 1 and2), nor in cells harvested after induction but lacking the coding sequence in the expression vector (lane 3). The N-terminal sequence (SAKPEVSLVR) of residues 1–10 of the purified protein was identical to that predicted for residues 2–11 of the human ODC (Fig. 1). About 35 mg of purified protein were obtained per liter of culture. Proteoliposomes reconstituted with recombinant ODC catalyzed a counter-exchange of external [14C]oxoglutarate for internal oxoglutarate with first order kinetics (rate constant 0.08 min−1), isotopic equilibrium being approached exponentially (data not shown). The exchange reaction was inhibited completely by a mixture of pyridoxal 5′-phosphate and bathophenanthroline. In the absence of substrate in the proteoliposomes, or if the solubilized protein was boiled before the incorporation into liposomes, there was no uptake of labeled external substrate. Similarly, no oxoglutarate/oxoglutarate exchange was detected by reconstitution of Sarkosyl-solubilized material from bacterial cells either lacking the expression vector for ODC or harvested immediately before induction of expression. Furthermore, the proteoliposomes did not catalyze homoexchange activities for phosphate, carnitine, glutamate, aspartate, glutamine, ornithine,l-malate, ADP, and ATP (internal concentration, 10 mm; external concentration, 1 mm). The substrate specificity of human ODC was investigated in greater detail by measuring the uptake of [14C]oxoglutarate into proteoliposomes that had been preloaded with a variety of substrates (Fig. 5 A) or the efflux of [14C]oxoglutarate from proteoliposomes in the presence of external nonradioactive substrates (Fig. 5 B). High [14C]oxoglutarate transport activities were observed when oxoglutarate, oxoadipate, glutarate, and adipate were used as counter-substrates on both sides of the liposomal membrane. High activities were also found with external pimelate and oxopimelate. To a lesser extent, internal pimelate, oxopimelate, and 2-aminopimelate, as well as both internal and external 2-aminoadipate, citrate, and oxaloacetate, also exchanged for labeled oxoglutarate. Low exchange, if any, was found with aspartate, fumarate, glutamate, glutathione, isocitrate, malate, maleate, malonate, oxalate, pyruvate, suberate, and succinate (Fig. 5, A and B). The uptake of 0.2 mm [14C]oxoglutarate by proteoliposomes containing 20 mm oxoglutarate (reaction time, 1 min) was inhibited almost completely by 2 mmpyridoxal 5′-phosphate (97%) and partly by 2 mmbathophenanthroline and 2 mm 2-cyanocynnamate (73 and 69% inhibition, respectively). Low concentrations of organic mercurials (10 μm) also inhibited markedly the ODC activity (p-chloromercuribenzene sulfonate, 58%; HgCl2, 64%; and p-hydroxymercuribenzoate, 47%). No significant inhibition was observed with 2 mm butylmalonate, phenylsuccinate, 1,2,3-benzenetricarboxylate, andN-ethylmaleimide (inhibitors of other characterized mitochondrial carriers) and 10 μm carboxyatractyloside (a powerful inhibitor of the ADP/ATP carrier). To obtain kinetic information about the [14C]oxoglutarate/oxoglutarate exchange, the dependence of the exchange rate on substrate concentration was investigated at different concentrations of externally added [14C]oxoglutarate (0.05–2 mm) at a constant internal concentration of 20 mm. The transport affinity (K m) and the specific activity (V max) values for oxoglutarate exchange at 25 °C, calculated from a standard double-reciprocal set of 35 experiments, were 0.22 ± 0.02 mm and 7.5 ± 1.2 mmol/min/g of protein, respectively. The activity was calculated by taking into account the amount of ODC recovered in the proteoliposomes after reconstitution. The inhibition constants (K i) of several externally added substrates are summarized in TableI. All of them increased the apparentK m without changing the V maxof [14C]oxoglutarate/oxoglutarate exchange (not shown) acting as competitive inhibitors. In general, the oxoglutarate/oxoglutarate exchange was prevented by external addition of each of the substrates that are transported by human ODC (Fig. 5,A and B), and it was not affected by substrates of other mitochondrial carriers such as phosphate, ADP, ornithine, and carnitine. However, suberate, which is not transported by the ODC protein, inhibited the exchange activity significantly (Table I), suggesting that it binds to the substrate binding site of ODC without being transported.Table IKi values for substrates competing with [14C]oxoglutarate uptakeSubstrateK imm2-Oxoadipate0.11 ± 0.01Adipate0.14 ± 0.01Pimelate0.40 ± 0.04Glutarate0.70 ± 0.072-Oxopimelate1.18 ± 0.12Suberate6.6 ± 0.8Citrate11.5 ± 1.32-Aminoadipate12.7 ± 1.5Oxaloacetate18.7 ± 2.2l-Malate>252-Aminopimelate>25The K i values were calculated from double-reciprocal plots of the rate of [14C]oxoglutarate/oxoglutarate exchange under variation of external oxoglutarate. Substrate concentrations were as follows: 20 mm internal 2-oxoglutarate and 0.05–2.0 mm external labeled 2-oxoglutarate. The competing substrates were added simultaneously with [14C]oxoglutarate at the appropriate concentrations. The values are averages ± S.D. of at least three experiments. Open table in a new tab The K i values were calculated from double-reciprocal plots of the rate of [14C]oxoglutarate/oxoglutarate exchange under variation of external oxoglutarate. Substrate concentrations were as follows: 20 mm internal 2-oxoglutarate and 0.05–2.0 mm external labeled 2-oxoglutarate. The competing substrates were added simultaneously with [14C]oxoglutarate at the appropriate concentrations. The values are averages ± S.D. of at least three experiments. The phylogenetic distance between S. cerevisiae and man frequently precludes the use of yeast sequences to identify human orthologs directly with certainty. The problem is even more severe when the protein in question is a member of a family with different functions but related sequences, as are the two isoforms of the yeast oxodicarboxylate carrier (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), which is a recently identified member of the family of mitochondrial transport proteins. Known family members are involved in the traffic of various substrates and metabolites across the inner membranes of the organelle. One possible solution, employed successfully in the cloning the rat dicarboxylate carrier (16Fiermonte G. Palmieri L. Dolce V. Lasorsa F.M. Palmieri F. Runswick M.J. Walker J.E. J. Biol. Chem. 1998; 273: 24754-24759Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), is to use the yeast sequence to identify orthologs in phylogenetically intermediate species where the genome sequence is known, such as C. elegans and D. melanogaster, and then to use these orthologs to identify potential orthologs in man. Thus, the yeast ODC isoforms were used to find clones in the C. elegans and D. melanogaster genomes, and they were used to identify a short related protein sequence in a human EST. This sequence proved to be part of the human ODC and provided the route to the complete human and rat ODC sequences. Both have the tripartite structure and the sequence motif that are characteristic of the mitochondrial carrier family (10Krämer R. Palmieri F. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers B. V., Amsterdam1992: 359-382Google Scholar, 11Walker J.E. Curr. Opin. Struct. Biol. 1992; 2: 519-526Crossref Scopus (105) Google Scholar, 12Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 13Palmieri F. van Ommen B. Papa S. Guerrieri F. Tager J.M. Frontiers of Cellular Bioenergetics. Kluwer Academic/Plenum Publishers, New York1999: 489-519Crossref Google Scholar). The transport characteristics of the human ODC are similar to the yeast ODC isoforms. The main differences are the ability of the human protein to transport 2-aminoadipate, although rather poorly, and its incapacity to transport l-malate. The properties of the human ODC differ markedly from those of the oxoglutarate-malate carrier, which has greatest affinities for C4 and C5 oxodicarboxylates and dicarboxylates (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar, 26Palmieri F. Quagliariello E. Klingenberger M. Eur. J. Biochem. 1972; 29: 408-416Crossref PubMed Scopus (104) Google Scholar, 27Bisaccia F. Indiveri C. Palmieri F. Biochim. Biophys. Acta. 1985; 810: 362-369Crossref PubMed Scopus (102) Google Scholar), whereas the human ODC prefers the C5-C7 homologs (Fig. 5 and Table I). In contrast to the bovine oxoglutarate-malate carrier, the human ODC does not transport malate, succinate, and maleate, but it does transport citrate, albeit at low efficiency. The low sequence identity between the ODC and oxoglutarate-malate carrier is also consistent with their different properties and functions. The best substrates for the human ODC, both on the external and the internal membrane surfaces, are 2-oxoadipate and 2-oxoglutarate. Therefore, the physiological role of the human ODC is most likely to be to catalyze the uptake of 2-oxoadipate into the mitochondrial matrix in exchange for internal 2-oxoglutarate, thus performing a central role in catabolism of lysine, hydroxylysine, and tryptophan. Since ODC functions by a counter-exchange mechanism, the carrier-mediated uptake of 2-oxoadipate requires the efflux of a counter-substrate. On the basis of transport measurements, 2-oxoglutarate may serve as the counter-substrate of ODC for 2-oxoadipate. The efflux of 2-oxoglutarate is required by the lysine-oxoglutarate reductase, which, in the first step of lysine catabolism, converts lysine and 2-oxoglutarate into saccharopine. In agreement with its central role in cell metabolism, the human ODC is expressed in all tissues that were analyzed. Another possible role for the human ODC may be to catalyze the uptake of 2-aminoadipate into the mitochondrial matrix when this amino acid is not rapidly transaminated to 2-oxoadipate in the cytosol, for example after diets rich in amino acids that cause a decrease in 2-oxoglutarate content in the cytosol and consequently an inhibition of the 2-aminoadipate aminotransferase. In this respect it is worth mentioning that transaminases interconverting oxoadipate and aminoadipate are present both in the cytosol and inside the mitochondria (28Tobes M.C. Mason M. J. Biol. Chem. 1977; 252: 4591-4599Abstract Full Text PDF PubMed Google Scholar, 29Takeuchi F. Otsuka H. Shibata Y. Biochim. Biophys. Acta. 1983; 743: 323-330Crossref PubMed Scopus (30) Google Scholar). The physiological role of ODC also suggests its possible involvement in 2-oxoadipate acidemia, which is accompanied by accumulation and excretion of large amounts of 2-oxoadipate, 2-aminoadipate and 2-hydroxyadipate in urine, and by the clinical symptoms of mental retardation, hypotonia, motor and developmental delay, cerebellar ataxia, and learning disability (1Przyrembel H. Bachmann D. Lombeck I. Becker K. Wendel U. Wadman S.K. Bremer H.J. Clin. Chim. Acta. 1975; 58: 257-269Crossref PubMed Scopus (55) Google Scholar, 2Wendel U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 3Wilson R.W. Wilson C.M. Gates S.C. Higgins J.V. Pediatr. Res. 1975; 9: 522-526Crossref PubMed Scopus (31) Google Scholar, 4Wilson R.W. Wilson C.M. Higgins J.V. Clin. Chim. Acta. 1976; 69: 323-332Crossref PubMed Scopus (9) Google Scholar, 5Fischer M.H. Brown R.R. Am. J. Med. Genet. 1980; 5: 35-41Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar, 7Vianey-Liaud C. Divry P. Cotte J. Teyssier G. J. Inherit. Metab. Dis. 1985; 8: 133-134Crossref PubMed Scopus (10) Google Scholar). The molecular defect(s) responsible for this disease have not been characterized. Fibroblasts of patients with this condition are unable to oxidize 2-amino[1-14C]adipic and 2-oxo[1-14C]adipic acid to 14CO2 (2Wendel U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar) to any significant extent. Therefore, it was suggested that the disease may be due to defective 2-oxoadipate dehydrogenase, but no such defect has ever been demonstrated. The alternative possibility that defective ODC might provide the basis for this human metabolic disease can now be investigated.