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Identification of the Yeast Mitochondrial Transporter for Oxaloacetate and Sulfate

1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês

10.1074/jbc.274.32.22184

1083-351X

Luigi Palmieri, Angelo Vozza, Gennaro Agrimi, Valeria De Marco, Michael J. Runswick, Ferdinando Palmieri, John E. Walker,

Metabolism and Genetic Disorders

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction inEscherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [35S]sulfate/sulfate and [35S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase. Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction inEscherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [35S]sulfate/sulfate and [35S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase. Saccharomyces cerevisiae has two principal pathways for replenishing the intermediates of the Krebs cycle that have been withdrawn for biosynthesis. They are the glyoxylate cycle and the carboxylation of pyruvate to oxaloacetate, catalyzed by two cytosolic isozymes of pyruvate carboxylase. (Mammalian pyruvate carboxylase is in the mitochondrial matrix.) These processes require traffic of substrates across the inner mitochondrial membrane. S. cerevisiae encodes 35 members of the mitochondrial carrier family (1Walker J.E. Runswick M.J. J. Bioenerg. Biomembr. 1993; 25: 435-446Crossref PubMed Scopus (196) Google Scholar, 2Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 3Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 4Palmieri F. van Ommen B. Papa S. Guerrieri F. Tager J.M. Frontiers in Cellular Bioenergetics. Kluwer Academic/Plenum Publishers, New York1999: 489-519Google Scholar), including the dicarboxylate and succinate-fumarate carriers (5Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (106) Google Scholar,6Palmieri L. Lasorsa F.M. De Palma A. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 417: 114-118Crossref PubMed Scopus (121) Google Scholar). The former catalyzes the import into mitochondria of succinate (or malate) in exchange for phosphate, producing a net uptake of succinate and supply of a substrate to the Krebs cycle (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar). The latter exchanges external succinate for fumarate and is required for gluconeogenesis (6Palmieri L. Lasorsa F.M. De Palma A. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 417: 114-118Crossref PubMed Scopus (121) Google Scholar,8Bojunga N. Kötter P. Entian K.-D. Mol. Gen. Genet. 1998; 260: 453-461Crossref PubMed Scopus (31) Google Scholar). Yeast gene disruption strains (Δ-DIC1 and Δ-SFC1) cannot grow on either ethanol or acetate as sole carbon source, but they grow well on glycerol, pyruvate and other nonfermentable substrates (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar, 9Fernandez M. Fernandez E. Rodicio R. Mol. Gen. Genet. 1994; 242: 727-735Crossref PubMed Scopus (35) Google Scholar). Growth of the Δ-DIC1strain on ethanol or acetate is restored by both low concentrations of oxaloacetate and other compounds that start the tricarboxylate cycle (and the oxidation of the acetyl-CoA unconsumed by the glyoxylate cycle) by generating either intramitochondrial oxaloacetate or other Krebs cycle intermediates (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar). Therefore, does oxaloacetate enter yeast mitochondria, and if so, how? The S. cerevisiae geneOAC1 1The name OAC1 has been reserved for the gene encoding the yeast oxaloacetate carrier. (formerly known as PMT or YKL120w) encodes a member of the mitochondrial carrier family of hitherto unknown function that is 29% identical to the yeast DIC, its closest relative in yeast and 30% identical to the bovine 2-oxoglutarate/malate carrier. OAC and the dicarboxylate carrier are the only carriers that cluster on a phylogenetic tree with the bovine oxoglutarate/malate carrier (10El Moualij B. Duyckaerts C. Lamotte-Brasseur J. Sluse F.E. Yeast. 1997; 13: 573-581Crossref PubMed Scopus (93) Google Scholar, 11Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (169) Google Scholar). Disruption of the OAC1 gene produced no phenotype on rich glycerol medium (12Colleaux L. Richard G.-F. Thierry A. Dujon B. Yeast. 1992; 8: 325-336Crossref PubMed Scopus (20) Google Scholar), and its transcript level was higher in synthetic than in rich medium (13Richard G.-F. Fairhead C. Dujon B. J. Mol. Biol. 1997; 268: 303-321Crossref PubMed Scopus (43) Google Scholar). We have overexpressed the OAC in E. coli, reconstituted it into phospholipid vesicles, and shown that it transports oxaloacetate, sulfate, and thiosulfate both in vitro and in vivo. One of its main functions is probably to carry oxaloacetate produced by cytoplasmic pyruvate carboxylase into the mitochondrial matrix. [2-l4C]Malonic acid, α-[1-l4C]ketoglutaric acid, [1,5-[14C] citric acid,l-[l4C(U)]glutamic acid,l-[l4C(U)]aspartic acid,l-[14C (U)] glutamine and [8-l4C]adenosine 5′-diphosphate trisodium salt were supplied by NEN Life Science Products. l-[1,4(2, 3)-l4C]Malic acid, [35S]sulfate, [32P]phosphate, andl-[methyl-3H]carnitine were obtained from Amersham Pharmacia Biotech, and [2,3-14C]fumaric acid andl-[2,3-3H]ornithine were from Sigma. Egg yolk phospholipids were purchased from Fluka, and cardiolipin andN-lauroylsarcosine were from Sigma. Amberlite XAD-2 was supplied by Supelco (Milan, Italy). The vector pYeDP60 was a generous gift of Dr. D. Pompon (Gif-sur-Yvette, France). Oligonucleotide primers were synthesized with sequences corresponding to the extremities of the coding sequence for OAC (nucleotides 216,990–217,964 on the positive strand of chromosome XI), with additional NdeI and HindIII sites. Other conditions have been given before (14Fiermonte 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 (116) Google Scholar). The overproduction of OAC as inclusion bodies in the bacterial cytosol and the purification of the inclusion bodies (5Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (106) Google Scholar, 15Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar) in host strain E. coli C0214(DE3) (14Fiermonte 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 (116) Google Scholar), the analysis of proteins by SDS-PAGE 2The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid) in 17.5% gels (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), and N-terminal sequencing (14Fiermonte 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 (116) Google Scholar) have been described previously. The yield of purified yeast protein/liter of bacterial culture was estimated by laser densitometry (14Fiermonte 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 (116) Google Scholar). The OAC was reconstituted into proteoliposomes as described for homologues (14Fiermonte 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 (116) Google Scholar, 17Fiermonte G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: 22782-22787Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). External substrate was removed from proteoliposomes on Sephadex G-75. Transport at 25 °C was started by adding [14C]malonate or [35S]sulfate to the proteoliposomes and terminated by addition of 0.l mm p-chloromercuribenzene sulfonate and 10 mm bathophenanthroline (the "inhibitor-stop" method (18Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar)). In controls, inhibitors were added with the labeled substrate. Finally, the external radioactivity was removed on Dowex AGl-X8, and the internal radioactivity was measured (18Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). The transport activity was the difference between experimental and control values. The initial rate of transport was calculated in μmol/min/g of protein from the time course of isotope equilibration (18Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). Various other transport activities were also assayed by inhibitor-stop. The OAC1 gene was deleted by homologous recombination of the auxotrophic marker URA3 at the OAC1 gene locus of S. cerevisiae strain YPH499. Its genotype is MATa ade2–101 his3-Δ 200 leu2-Δ1 ura3–52 trpl-Δ 63lys2-801 OAC1::URA3. The coding sequence was cloned into the yeast expression plasmid pYeDP60 (19Pompon D. Louerat B. Bronine A. Urban P. Methods Enzymol. 1996; 272: 51-64Crossref PubMed Google Scholar) yielding plasmidOAC1-pYeDP60. It was introduced into the Δ-OAC1yeast strain, and transformants were selected for adenine auxotrophy. Wild-type fungi and the deleted strain were grown either in rich medium containing 2% bactopeptone and 1% yeast extract (YP) or in synthetic complete medium (20Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar) supplemented with either fermentatable or nonfermentable carbon sources (2% glucose or 2% galactose, and 3% glycerol, 2% ethanol, and 3% acetate or 2% lactate). The Δ-OAC1 strain transformed with the OAC1-pYeDP60 construct (the OAC1-pYeDP60 strain) was precultured in synthetic complete medium lacking uracil, supplemented with 2% ethanol. For preparation of mitochondria, the preculture was diluted 35-fold in YP medium. The Gal-Cyc promoter was repressed by addition of 0.1% glucose. The OAC1-pYeDP60 cells were grown to exponential phase, and galactose (0.4%) was added 6 h before harvesting. Yeast was grown in YP broth supplemented with 2% ethanol, and mitochondria were isolated (21Daum G. Gasser S.M. Schatz G. J. Biol. Chem. 1982; 257: 13075-13080Abstract Full Text PDF PubMed Google Scholar). Integral membrane proteins were separated from soluble and peripheral proteins by extraction of mitochondria (1 mg of protein/ml) with 0.1 m sodium carbonate for 30 min at 0 °C and centrifugation (226,000 × g for 1 h at 2 °C). The pellet contained integral membrane proteins, and the supernatant contained peripheral and soluble proteins (22Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar). Mitochondria were resuspended in buffer (10 mmK+-MOPS, pH 7.2, 0.25 m sucrose, 5 mm EDTA, 1 mm o-phenanthroline, and 0.2 m KCl; final protein concentration, 1 mg/ml) and extracted with digitonin (23Hartl F.-U. Ostermann J. Guiard B. Neupert W. Cell. 1987; 51: 1027-1037Abstract Full Text PDF PubMed Scopus (180) Google Scholar). The solutions were mixed and placed on ice for 1 min. They were diluted with 4 volumes of buffer and centrifuged (100,000 × g for 10 min). The soluble proteins were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE and Western blotting with a rabbit antibody against recombinant OAC (24Dietmeier K. Zara V. Palmisano A. Palmieri F. Voos W. Schlossmann J. Moczko M. Kispal G. Pfanner N. J. Biol. Chem. 1993; 268: 25958-25964Abstract Full Text PDF PubMed Google Scholar). Swelling of mitochondria in an isoosmotic solution of an ammonium salt indicates that an anion permeates the mitochondrion with protons (or in exchange for hydroxyl ions) (25Chappell J.B. Br. Med. Bull. 1968; 24: 150-157Crossref PubMed Scopus (280) Google Scholar). Molecular ammonia is protonated inside the mitochondria, and the pH gradient collapses. The rate of swelling of mitochondria was monitored by the decrease in A 546 (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar). Yeast mitochondria (100 μg of protein) were added to 1 ml of various ammonium salts (120 mm), 20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 5 μm rotenone, and 0.1 μm antimycin. The membrane potential of mitochondria was assessed from the fluorescence changes of the voltage-sensitive dye DiSC3(5) (26Zara V. Dietmeier K. Palmisano A. Vozza A. Rassow J. Palmieri F. Pfanner N. Mol. Cell. Biol. 1996; 16: 6524-6531Crossref PubMed Scopus (27) Google Scholar). Yeast OAC was overexpressed inE. coli C0214(DE3) at high levels (Fig.1, lane 4). Its apparent molecular mass was about 37 kDa (calculated value including the initiator methionine, 35,165 Da). The purified inclusion bodies gave rise a single band on SDS-PAGE gels with the same apparent molecular mass (Fig. 1, lane 5). It was detected neither in bacteria harvested immediately before induction of expression (lane 2) nor in cells harvested after induction but lacking the OAC coding sequence in the expression vector (lane 3). The N-terminal sequence of the 37-kDa protein, SSDNSKQDKQIE, is identical to residues 2–13 of OAC. About 60 mg of purified protein (Fig. 1, lane 5) were obtained per liter of culture. The reconstituted protein catalyzed an active [l4C]malonate/malonate exchange and a more active [35S]sulfate/sulfate exchange (TableI) that were inhibited by a mixture ofp-chloromercuribenzene sulfonate and bathophenanthroline. Homoexchange activities more than 1 order of magnitude lower than these activities were measured for malate, oxoglutarate, and phosphate. No homoexchange activities were detected with OAC that had been boiled before incorporation into liposomes or by reconstitution of sarkosyl-solubilized material from bacterial cells either lacking the expression vector for OAC or harvested immediately before induction of overexpression. The proteoliposomes did not catalyze homoexchanges for fumarate, citrate, glutamate, aspartate, glutamine, carnitine, ornithine, and ADP (external concentration, 1 mm; internal concentration, 10 mm) (Table I).Table IRates of homo-exchanges of various substrates in proteoliposomes containing recombinant yeast OACSubstrate transportμmol/min/g proteinMalonate222l-Malate25Fumarate0Sulfate596Phosphate10Oxoglutarate12Citrate0Glutamate0Aspartate0Glutamine0Ornithine0Carnitine0ADP0Transport was initiated by adding radioactive substrate (final concentration, 1 mm) to proteoliposomes preloaded internally with the same substrate (concentration, 10 mm). Similar results were obtained in three independent experiments. Open table in a new tab Transport was initiated by adding radioactive substrate (final concentration, 1 mm) to proteoliposomes preloaded internally with the same substrate (concentration, 10 mm). Similar results were obtained in three independent experiments. In Fig. 2 A, the time-courses are compared of uptake by proteoliposomes of 1 mm[l4C]malonate measured either as uniport (in the absence of internal malonate) or as exchange (in the presence of 10 mm malonate). Both data sets fitted a first-order rate equation with rate constants (k) for the exchange and the uniport reactions of 0.15 and 0.14 min−1, respectively. Maximum uptake was approached after 30 min. The corresponding values at infinite time were 1.46 and 0.16 μmol/mg. The ratio of maximal substrate uptake by the exchange and by the uniport was 9.1, in 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 malonate uptake (the product of k and intraliposomal quantity of malonate taken up at equilibrium (18Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar)) were 213 and 23 μmol/min/g of protein for the exchange and uniport reactions, respectively. The addition of 10 mm unlabeled malonate after 30 min incubation, when radioactive uptake by the proteoliposomes had approached equilibrium, caused an extensive efflux of radiolabeled malonate from both malonate-loaded and unloaded proteoliposomes (Fig. 2 A). This efflux indicates that the [l4C]malonate taken up by uniport is released by exchange for externally added substrate. Upon addition to liposomes that had been preincubated for 30 min with 1 mm [l4C]malonate in the absence of internal substrate of 10 mm malonate, oxaloacetate, or sulfate, an extensive efflux of intraliposomal radioactivity was observed (Fig.2 B). Upon addition of malate, oxoglutarate, and phosphate, very little efflux was detected, and addition of fumarate had no effect. These results indicate clearly that oxaloacetate, sulfate, and malonate are good substrates for reconstituted OAC. The substrate specificity of reconstituted OAC was investigated further by measuring the uptake of [l4C]malonate and [35S]sulfate into proteoliposomes that had been preloaded with various substrates (Table II). The highest activities were observed in the presence of internal sulfate, thiosulfate, oxaloacetate, and malonate. Lower activity was found in the presence of internal malate, succinate, oxoglutarate, and phosphate. Virtually no exchange was detected with internal fumarate, pyruvate, aspartate, glutamate, citrate, ADP, ornithine, glutamine, and carnitine. The residual activity in the presence of these substrates was virtually the same as the activity observed without internal substrate. Therefore, the substrate specificity of the OAC protein is confined essentially to sulfate, thiosulfate, and the physiologically important dicarboxylate oxaloacetate.Table IIDependence on internal substrate of the transport properties of proteoliposomes containing recombinant yeast OACInternal substrate (10 mm)Substrate transport[14C]Malonate[35S]Sulfateμmol/5 min/g proteinNone (Cl−present)129116Malonate8831252Sulfate9471597Thiosulfate9641488Oxaloacetate9561305l-Malate296621Succinate214263Oxoglutarate258407Phosphate253352Fumarate145198Pyruvate123107Aspartate92104Glutamate10598Citrate137152ADP114125Ornithine138146Glutamine127109Carnitine130182Proteoliposomes were preloaded internally with various substrates (concentration, 10 mm). Transport was started by the external addition of [14C]malonate or [35S]sulfate (final concentrations, 0.5 mm) and terminated after 5 min. Similar results were obtained in three independent experiments. Open table in a new tab Proteoliposomes were preloaded internally with various substrates (concentration, 10 mm). Transport was started by the external addition of [14C]malonate or [35S]sulfate (final concentrations, 0.5 mm) and terminated after 5 min. Similar results were obtained in three independent experiments. The uptake of [l4C]malonate by proteoliposomes was inhibited by sulfydryl reagents (mersalyl,p-chloromercuribenzene sulfonate, andN-ethylmaleimide), by pyridoxal 5-phosphate, and by bathophenanthroline (Table III). The impermeable dicarboxylate analogues butylmalonate and benzylmalonate also inhibited the transport activity strongly. In contrast, carboxyatractyloside, 1,2,3-benzenetricarboxylate and α-cyanocinnamate, inhibitors of other characterized mitochondrial carriers, had no effect on the activities of reconstituted OAC. The ability of nonradioactive potential substrates to inhibit the uptake of [l4C]malonate was also examined (Table III). Malonate transport was prevented by external addition of malonate, sulfate, thiosulfate, and oxaloacetate, substrates that are transported by OAC (Tables I and II and Fig. 2 B). To a lesser extent, poor substrates such as malate, phosphate, oxoglutarate, and succinate inhibited the OAC-catalyzed transport of [l4C]malonate.Table IIIEffect of inhibitors and externally added substrates on the uptake of [ 14C]malonate into proteoliposomes reconstituted with recombinant OACReagentsInhibition%Experiment 1Mersalyl99p-Chloromercuriphenylsulfonate99N-Ethylmaleimide68Bathophenanthroline85Pyridoxal 5′-phosphate84Butylmalonate93Benzylmalonate881,2,3-Benzenetricarboxylate12Carboxyatractyloside4α-Cyanocinnamate7Experiment 2Malonate96Sulfate87Thiosulfate82Sulfite85Oxaloacetate84Malate56Phosphate46Oxoglutarate30Succinate23Pyruvate6Citrate7ADP4Carnitine10Ornithine7Transport was started by adding [14C]malonate (final concentration, 0.1 mm) to proteoliposomes containing 10 mm NaCl and no substrate. Thiol reagents, pyridoxal 5′-phosphate, carboxyatractyloside and α-cyanocinnamate were added 3 min before the labeled substrate; the other inhibitors and external substrates were added together with [14C]malonate. All inhibitors and substrates were present at final concentrations of 2 mm, except for organic mercurials (0.001 mm), carboxyatractyloside and α-cyanocinnamate (0.1 mm), and pyridoxal 5′-phosphate (10 mm). The control values for uninhibited malonate uptake were 12.4 (experiment 1) and 14.0 (experiment 2) μmol/min per g of protein. Similar results were obtained in three independent experiments. Open table in a new tab Transport was started by adding [14C]malonate (final concentration, 0.1 mm) to proteoliposomes containing 10 mm NaCl and no substrate. Thiol reagents, pyridoxal 5′-phosphate, carboxyatractyloside and α-cyanocinnamate were added 3 min before the labeled substrate; the other inhibitors and external substrates were added together with [14C]malonate. All inhibitors and substrates were present at final concentrations of 2 mm, except for organic mercurials (0.001 mm), carboxyatractyloside and α-cyanocinnamate (0.1 mm), and pyridoxal 5′-phosphate (10 mm). The control values for uninhibited malonate uptake were 12.4 (experiment 1) and 14.0 (experiment 2) μmol/min per g of protein. Similar results were obtained in three independent experiments. TheK m and V max values for malonate uptake by unloaded proteoliposomes (measured as uniport at 25 °C), from a typical experiment (Fig.3) were 0.11 mm and 25 μmol/min/g of protein, respectively. The average values ofK m and V max from 18 experiments were 0.1 ± 0.01 mm and 22 ± 6 μmol/min/g of protein, respectively. Oxaloacetate and sulfate inhibited malonate uptake competitively (Fig. 3). All of the compounds summarized in Table IV are competitive inhibitors with respect to malonate, because they were found to increase the apparent K m without changing theV max of malonate uptake (not shown). TheK i value of l-malate is significantly lower than the respective K m value (> 6 mm) as determined from homoexchange rate measurements, indicating that although malate is poorly transported by OAC, it has a high affinity for its substrate-binding site.Table IVCompetition with [ 14C]malonate uptake in proteoliposomes containing recombinant yeast OACSubstrateK immSulfate0.07 ± 0.02Thiosulfate0.17 ± 0.02Sulfite0.06 ± 0.01Oxaloacetate0.25 ± 0.09Malate1.1 ± 0.2Oxoglutarate3.4 ± 0.4Phosphate2.0 ± 0.4The values were calculated from double reciprocal plots of the rate of [14C]malonate uptake versus substrate concentrations. For experimental conditions, see Fig. 3. The competing substrates were added at the appropriate concentrations simultaneously with [14C]malonate. The data represent the means ± S.D. of at least three different experiments. Open table in a new tab The values were calculated from double reciprocal plots of the rate of [14C]malonate uptake versus substrate concentrations. For experimental conditions, see Fig. 3. The competing substrates were added at the appropriate concentrations simultaneously with [14C]malonate. The data represent the means ± S.D. of at least three different experiments. A single immunoreactive band with an apparent molecular mass of about 36.2 kDa was detected in the wild-type mitochondria, but not in mitochondria from the Δ-OAC1 strain (Fig. 4). The calculated mass of OAC including the initiator methionine is 35,165 Da. The contents of succinate-fumarate, ADP/ATP, phosphate, and the dicarboxylate carriers detected with specific antibodies were essentially the same in wild-type and Δ-OAC1 mitochondria. Therefore, the absence of OAC from the Δ-OAC1 strain does not reduce expression of other carriers. The submitochondrial location of OAC was examined by separation of soluble and peripheral proteins from integral membrane proteins of wild-type mitochondria by carbonate treatment (Fig.5, A and B). OAC remained in the membrane protein fraction, as did the ADP/ATP carrier and Tom40 (marker proteins of inner and outer mitochondrial membranes, respectively), but the intermembrane space protein cytochrome b2 and the matrix protein hsp70 were in the soluble and peripheral protein fraction. Therefore, OAC is an integral mitochondrial membrane protein. Both OAC and the ADP/ATP carrier were solubilized from wild-type mitochondria at the same concentration of digitonin, which was greater than that required to solubilize the outer membrane protein Tom40 (Fig.5 C). Therefore, OAC is an integral protein of the inner mitochondrial membrane. Wild-type and Δ-OAC1cells had similar growth characteristics in complete synthetic medium and in YP broth, but their mitochondria differed in several respects. For example, wild-type mitochondria swelled in 120 mmsolutions of ammonium salts of malonate, oxaloacetate, sulfate, and thiosulfate (Fig. 6), whereas mitochondria from the Δ-OAC1 strain did not do so appreciably. Both mitochondria swelled in ammonium phosphate, indicating the presence of an active phosphate carrier, but neither did in the ammonium salts of malate (mainly transported by the dicarboxylate carrier; swelling requires a catalytic trace of phosphate), fumarate (transported by the succinate-fumarate carrier), or chloride (impermeant anion). Therefore, the absence of OAC does not affect the integrity of the mitochondria and the activity of other carriers. The validity of the swelling method was confirmed by control experiments on mitochondria from a strain in which the gene for the dicarboxylate carrier has been disrupted (ΔDIC1). They swelled in the presence of the ammonium salts of low affinity substrates of the dicarboxylate carrier (oxaloacetate, malonate, sulfate, and thiosulfate) at a very similar rate to wild-type mitochondria (Fig. 6), but not in the presence of ammonium malate plus phosphate, in agreement with the absence of the dicarboxylate carrier (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar). When the external pH was decreased from 8.0–5.9, the rate of swelling in ammonium sulfate increased strongly (Fig.7 A), confirming the dependence of the sulfate/H+ influx on ΔpH. Similar results were obtained with ammonium oxaloacetate and ammonium malonate (data not shown). Further evidence that the influx into the mitochondrial matrix of sulfate, oxaloacetate, and malonate is carrier-mediated came from the study of the effects of inhibitors of OAC on swelling. In isoosmotic ammonium sulfate, swelling of wild-type mitochondria is strongly inhibited by 20 mm bathophenanthroline and abolished almost completely by 0.2 mm mersalyl orp-chloromercuribenzenesulfonic acid (Fig. 7 B). Organic mercurials and bathophenanthroline inhibited mitochondrial swelling in ammonium salts of oxaloacetate and malonate (data not shown). Therefore, mitochondrial swelling and activity of recombinant OAC are inhibited by the same reagents. Finally, the membrane potential (Δψ) of wild-type and Δ-OAC1 mitochondria were compared with the fluorescence dye DiSC3(5) (27Sims P.J. Waggoner A.S. Wang C.-H. Hoffmann J.F. Biochemistry. 1974; 13: 3315-3330Crossref PubMed Scopus (764) Google Scholar). The fluorescence difference of the mitochondria and substrates, before and after the addition of valinomycin (in the presence of K+), was virtually the same in wild-type and Δ-OAC1 mitochondria (data not shown). Therefore, deletion of the OAC1 gene does not influence the coupling properties of the inner mitochondrial membrane. OAC was expressed about 30 times higher in mitochondria in theOAC1-pYeDP60 strain than in wild-type mitochondria. Increased activity was evident from rapid mitochondrial swelling in ammonium sulfate or ammonium malonate (Fig. 6), and from increased uptake into proteoliposomes reconstituted with a digitonin extract of those mitochondria of [35S]sulfate in exchange for sulfate, oxaloacetate, or malonate (Fig.8). The sulfate/sulfate, sulfate/oxaloacetate, and sulfate/malonate exchange activities were several times higher than in Δ-OAC1 and wild-type mitochondria, whereas the sulfate/phosphate exchange (mainly the dicarboxylate carrier) was little increased in OAC1-pYeDP60 mitochondria. Similarly, the [l4C]malate/phosphate exchange (the defining reaction of the dicarboxylate carrier) was virtually the same in wild-type and OAC1-pYeDP60 mitochondria (not shown). Therefore, the large difference between the activities of the reactions catalyzed by OAC in Δ-OAC1 and OAC1-pYeDP60 mitochondria strongly supports the conclusion that OAC functions as a transporter for oxaloacetate and sulfate. The identification of yeast OAC as an oxaloacetate and sulfate carrier provides a further demonstration of the utility of the procedure for overexpressing the mitochondrial carriers in E. coli and of reconstituting the recombinant proteins in liposomes in order to study their transport properties. This procedure, originally developed with the bovine oxoglutarate/malate carrier (15Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar), has been applied subsequently to the independently identified phosphate (17Fiermonte G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: 22782-22787Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 28Wohlrab H. Briggs C. Biochemistry. 1994; 33: 9371-9375Crossref PubMed Scopus (53) Google Scholar) and carnitine (29Indiveri C. Iacobazzi V. Giangregorio N. Palmieri F. Biochem. Biophys. Res. Commun. 1998; 249: 589-594Crossref PubMed Scopus (72) Google Scholar) carriers and has allowed the yeast carriers for citrate (30Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), dicarboxylate (5Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (106) Google Scholar), ornithine (31Palmieri 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), and succinate-fumarate (6Palmieri L. Lasorsa F.M. De Palma A. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 417: 114-118Crossref PubMed Scopus (121) Google Scholar) to be identified. The use of S. cerevisiae strains containing deleted carrier genes has provided evidence that the dicarboxylate and ornithine carriers operate with the same transport properties in vivoas in vitro (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar, 32Soetens O. Crabeel M. El Moualij B. Duyckaerts C. Sluse F. Eur. J. Biochem. 1998; 258: 702-709Crossref PubMed Scopus (26) Google Scholar). Four observations demonstrate that reconstituted OAC catalyzes both exchange and uniport. First, the ratio between malonate taken up by exchange and uniport at equilibrium agrees with the expected value if all of the carrier-loaded liposomes catalyze both exchange and uniport of malonate. Second, addition of unlabeled substrates releases nearly all [14C]malonate taken up by uniport. Third, both exchange and uniport are inhibited by low concentrations of bathophenanthroline and mercurials. Fourth, the first-order rate constant for uniport is practically the same as that measured for the exchange. However, the exchange rate is much faster than the uniport rate, and so the OAC resembles other reconstituted mitochondrial carriers (phosphate, carnitine, and ornithine) (31Palmieri 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, 33Stappen R. Krämer R. Biochim. Biophys. Acta. 1993; 1149: 40-48Crossref PubMed Scopus (50) Google Scholar,34Indiveri C. Tonazzi A. Palmieri F. Biochim. Biophys. Acta. 1991; 1069: 110-116Crossref PubMed Scopus (67) Google Scholar). The physiologically important reaction for the phosphate carrier is the uniport (Pi/H+ symport), whereas for the carnitine and the ornithine carriers, both exchange and unidirectional reactions are important. Because the apparentV max depends on the experimental conditions of the reconstituted system, and the amount of active carrier molecules present in the proteoliposomes is not known, the transport rates measured in the reconstituted system may differ substantially from the activities in vivo. The substrate specificity of the OAC is distinct from those of the dicarboxylate and oxoglutarate carriers (the principal substrates of which are malate and phosphate, and oxoglutarate and malate, respectively), or from any other characterized mitochondrial carrier. Nonetheless, l-malate, phosphate, and oxoglutarate are transported to some extent by the OAC protein, and so its specificity overlaps with those of the dicarboxylate and the oxoglutarate carriers. When homoexchanges are measured (Table I), the rate is less than 120 of that of the sulfate/sulfate exchange. However, a low rate of transport takes place when the exchange of intraliposomal malate, oxoglutarate, or phosphate at high concentrations (10 mm) is measured against sulfate and malonate, which are good substrates for OAC (Table II). The inhibition constants of oxoglutarate, phosphate, and expecially malate on the rate of malonate uptake (Table III) also indicate a rather significant affinity for OAC. However, the half-saturation transport constant for malate (>6 mm) is much higher than its K i, clearly showing that malate binds to the substrate-binding site of OAC, although it is poorly transported. Given the rather close similarity of the sequences of the OAC and the dicarboxylate and the oxoglutarate carriers, this is expected. The swelling properties of the wild-type yeast mitochondria were strikingly different from those of rat liver. The former swell in ammonium oxaloacetate, sulfate, thiosulfate, and malonate, whereas the latter do not swell either in ammonium oxaloacetate, 3L. Palmieri, A. Vozza, G. Agrimi, V. De Marco, M. J. Runswick, F. Palmieri, and J. E. Walker, unpublished observations. or in ammonium sulfate, thiosulfate, or malonate (35Crompton M. Palmieri F. Capano M. Quagliariello E. Biochem. J. 1974; 142: 127-137Crossref PubMed Scopus (47) Google Scholar, 36Crompton M. Palmieri F. Capano M. Quagliariello E. FEBS Lett. 1974; 46: 247-250Crossref PubMed Scopus (33) Google Scholar). Therefore, oxaloacetate, sulfate, thiosulfate, and malonate are transported into the yeast mitochondrial matrix with protons (or in exchange for hydroxyl ion) (25Chappell J.B. Br. Med. Bull. 1968; 24: 150-157Crossref PubMed Scopus (280) Google Scholar), and the ΔpH component of the protonmotive force drives their entry into the matrix (37Mitchell P. Nature. 1961; 191: 144-148Crossref PubMed Scopus (2924) Google Scholar). This transport mechanism is consistent with the ability of the recombinant OAC to catalyze influx of its substrates into proteoliposomes in the absence of internal substrate. As the S. cerevisiaepyruvate carboxylase is cytoplasmic, one physiological role of OAC is probably to catalyze uptake of oxaloacetate into mitochondria, a role supported by the higher transcript level in synthetic medium than in rich medium (13Richard G.-F. Fairhead C. Dujon B. J. Mol. Biol. 1997; 268: 303-321Crossref PubMed Scopus (43) Google Scholar). The OAC1 gene is not essential for growth of S. cerevisiae, and it is likely that in the absence of OAC, oxaloacetate is converted to malate in the cytoplasm, and malate enters the mitochondrion via the dicarboxylate carrier in exchange for phosphate. A role in anaplerosis for both the OAC and the dicarboxylate carrier (7Palmieri L. Vozza A. Hönlinger A. Dietmeier K. Palmisano A. Zara V. Palmieri F. Mol. Microbiol. 1999; 31: 569-577Crossref PubMed Scopus (75) Google Scholar) is consistent with the failure of a yeast strain with both carrier genes deleted to grow on nonfermentable carbon sources (not shown). Another possible role for the OAC may be to transfer reducing equivalents from the mitochondrial matrix to the cytosol by catalyzing a malate/oxaloacetate exchange when the intramitochondrial concentrations of NADH and malate are high. A malate-oxaloacetate shuttle has been proposed to operate in mammalian and plant mitochondria (38Gimpel J.A. De Haan E.J. Tager J.M. Biochim. Biophys. Acta. 1973; 292: 582-591Crossref PubMed Scopus (60) Google Scholar, 39Passarella S. Palmieri F. Quagliariello E. Arch. Biochem. Biophys. 1977; 180: 160-168Crossref PubMed Scopus (36) Google Scholar, 40Hanning I. Baumgarten K. Schott K. Heldt H.W. Plant Physiol. 1999; 119: 1025-1031Crossref PubMed Scopus (43) Google Scholar). The OAC and the dicarboxylate carrier are the only proteins with significant sequence homology to the bovine oxoglutarate carrier, and so it is unlikely that S. cerevisiae has an oxoglutarate/malate carrier. One important difference between animal and yeast mitochondria is the mechanism for reoxidation of cytosolic NADH. In animals, the oxoglutarate/malate and glutamate/aspartate carriers produce a net transport of reducing equivalents from the cytosol to the mitochondrial matrix by the aspartate-malate shuttle. InS. cerevisiae and in plants, cytosolic NADH is oxidized on the exterior of the inner mitochondrial membrane, making the presence of the oxoglutarate/malate carrier unnecessary. Therefore, it is possible that OAC and the oxoglutarate carrier have evolved from a close common ancestor. Transport of sulfate and thiosulfate may be important in sulfur metabolism. In mammals, the conversion of thiosulfate to sulfite is catalyzed by mitochondrial thiosulfate sulfurtransferase (rhodanase) and thiosulfate reductase (41De Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2567) Google Scholar, 42Koj A. Frendo J. Janik Z. Biochem. J. 1967; 103: 791-795Crossref PubMed Scopus (52) Google Scholar). A "sulfate uniport by a transport system sensitive to N-ethylmaleimide that shares the properties of the phosphate carrier" (i.e. electroneutral H+-compensated) in rat mitochondria (43Saris N.-E.L. Biochem. J. 1980; 192: 911-917Crossref PubMed Scopus (11) Google Scholar) is the only current evidence for a mammalian OAC.