Identification of the Mitochondrial ATP-Mg/Pi Transporter
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m400445200
ISSN1083-351X
AutoresGiuseppe Fiermonte, Francesco De Leonardis, Simona Todisco, Luigi Palmieri, Francesco M. Lasorsa, Ferdinando Palmieri,
Tópico(s)RNA modifications and cancer
ResumoThe mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66–75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca2+-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/Pi carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/Pi carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations. The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66–75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca2+-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/Pi carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/Pi carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations. Functional studies in intact mitochondria have indicated that the inner membranes of mitochondria contain a variety of proteins that transport important metabolites, nucleotides, and cofactors into and out of the matrix. Family members have three tandem repeated sequences, each of ∼100 amino acids, made of two hydrophobic transmembrane α-helices joined by a hydrophilic loop. The tandem repeats contain conserved features. So far, 18 members of the family have been identified. They are the uncoupling protein; the carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, dicarboxylates, ornithine, succinate-fumarate, oxalacetate-sulfate, oxodicarboxylates, deoxynucleotides, aspartateglutamate, glutamate, thiamine pyrophosphate, and S-adenosylmethionine; the adenine nucleotide transporter in peroxisomes; and the dicarboxylate-tricarboxylate carrier in plants (see Refs. 1Palmieri F. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 689-709Crossref PubMed Scopus (637) Google Scholar, 2Palmieri L. Lasorsa F.M. Vozza A. Agrimi G. Fiermonte G. Runswick M.J. Walker J.E. Palmieri F. Biochim. Biophys. Acta. 2000; 1459: 363-369Crossref PubMed Scopus (83) Google Scholar, 3Picault N. Hodges M. Palmieri L. Palmieri F. Trends Plant Sci. 2004; 9: 138-146Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar for reviews and Refs. 4Marobbio C.M.T. Vozza A. Harding M. Bisaccia F. Palmieri F. Walker J.E. EMBO J. 2002; 21: 5653-5661Crossref PubMed Scopus (88) Google Scholar and 5Marobbio C.M.T. Agrimi G. Lasorsa F.M. Palmieri F. EMBO J. 2003; 22: 5975-5982Crossref PubMed Scopus (97) Google Scholar). The functions of other family members found in genome sequences are unknown. At the same time, there are other transport activities observed in intact mitochondria that have yet to be associated with specific proteins. An example is the net transport of adenine nucleotides across the inner mitochondrial membrane (see Ref. 6Aprille J.R. Fisskum G. Mitochondrial Physiology and Pathology. Van Nostrand Reinhold Co. Inc., New York1986: 66-99Google Scholar for review), which is essential for modulation of the matrix adenine nucleotide concentration that is subject to change in many physiopathological situations (see Refs. 7Aprille J.R. FASEB J. 1988; 2: 2547-2556Crossref PubMed Scopus (94) Google Scholar and 8Aprille J.R. Lemaster J.J. Hackenbrock C.R. Thurman R.G. Westerhoff H.V. Integration of Mitochondrial Function. Plenum Press, New York1988: 393-404Crossref Google Scholar for reviews). To explain this activity, in the early 1980s, a new mitochondrial carrier was postulated (9Pollak J.K. Sutton R. Biochem. J. 1980; 192: 75-83Crossref PubMed Scopus (29) Google Scholar, 10Aprille J.R. Asimakis G.K. Arch. Biochem. Biophys. 1980; 201: 564-575Crossref PubMed Scopus (117) Google Scholar, 11Austin J. Aprille J.R. J. Biol. Chem. 1984; 259: 154-160Abstract Full Text PDF PubMed Google Scholar), the ATP-Mg/Pi carrier, whose properties have been investigated thoroughly in isolated mitochondria (9Pollak J.K. Sutton R. Biochem. J. 1980; 192: 75-83Crossref PubMed Scopus (29) Google Scholar, 10Aprille J.R. Asimakis G.K. Arch. Biochem. Biophys. 1980; 201: 564-575Crossref PubMed Scopus (117) Google Scholar, 11Austin J. Aprille J.R. J. Biol. Chem. 1984; 259: 154-160Abstract Full Text PDF PubMed Google Scholar, 12Austin J. Aprille J.R. Arch. Biochem. Biophys. 1983; 222: 321-325Crossref PubMed Scopus (27) Google Scholar, 13Haynes Jr., R.C. Picking R.A. Zaks W.J. J. Biol. Chem. 1986; 261: 16121-16125Abstract Full Text PDF PubMed Google Scholar, 14Nosek M.T. Dransfield D.T. Aprille J.R. J. Biol. Chem. 1990; 265: 8444-8450Abstract Full Text PDF PubMed Google Scholar, 15Joyal J.L. Aprille J.R. J. Biol. Chem. 1992; 267: 19198-19203Abstract Full Text PDF PubMed Google Scholar, 16Nosek M.T. Aprille J.R. Arch. Biochem. Biophys. 1992; 296: 691-697Crossref PubMed Scopus (23) Google Scholar, 17Dransfield D.T. Aprille J.R. Am. J. Physiol. 1993; 264: C663-C670Crossref PubMed Google Scholar, 18Hagen T. Joyal J.L. Henke W. Aprille J.R. Arch. Biochem. Biophys. 1993; 303: 195-207Crossref PubMed Scopus (12) Google Scholar). This carrier catalyzes a reversible counterexchange of ATP-Mg for Pi that accounts for the net uptake or efflux of ATP-Mg as Pi recycles rapidly through the membrane via the phosphate carrier. The carrier-mediated transport consists of an electroneutral divalent exchange between ATP-Mg2- and HPO42− as shown by the equilibrium ratios of these substrates under every condition tested and by the ability of monofluorophosphate (but not difluorophosphate) to exchange for ATP-Mg. ADP can also be transported by the ATP-Mg/Pi carrier, and nonproductive exchanges (ATP-Mg2- for ATP-Mg2- and HPO42− for HPO42−) are also catalyzed by the carrier. The transport activity is insensitive to carboxyatractyloside and inhibited by mersalyl. Furthermore, the ATP-Mg/Pi carrier is saturable and activated by calcium. However, its activity is slow compared with that of the reactions for ATP synthesis and hydrolysis and with that of most other mitochondrial carriers. Consequently, the ATP transported by this carrier distributes quickly into the ATP, ADP, and (eventually) AMP pools, and the net transport of ATP-Mg results in a change in the adenine nucleotide concentrations without changing the ATP/ADP ratios in the cytosol and mitochondrial matrix compartments. In this study, the identification of three human isoforms of the mitochondrial ATP-Mg/Pi carrier (APC) 1The abbreviations used are: APC, ATP-Mg/Pi carrier; EST, expressed sequence tag; AAC, ADP/ATP carrier; CTD, C-terminal domain; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; BFP, blue fluorescent protein. is described. By using the ADP/ATP carrier sequence, human expressed sequence tags (ESTs) that encode fragments of the three related proteins were identified. They provided information to complete the human cDNA sequences. The encoded proteins are 458, 468, and 489 amino acids long and ∼60% identical in sequence and were found to be localized to mitochondria. They were overexpressed in Escherichia coli, purified, reconstituted into phospholipid vesicles, and shown to be the human isoforms of the ATP-Mg/Pi carrier. The main function of these proteins is probably to catalyze the net uptake or efflux of adenine nucleotides into or from the mitochondria. This work presents the first information on the molecular properties of the mitochondrial carriers responsible for the ATP-Mg/Pi exchange and a definitive identification of their genes. Sequence Search and Analysis—Human EST and genome data bases 2Available at www.ebi.ac.uk, ensembl.ebi.ac.uk, and www.ncbi.nlm.nih.gov. were screened with the protein sequence of human AAC1 (GenBank™/EBI accession number NM_001151) and with the protein sequences of APC1, APC2, and APC3, respectively, using TBLASTN. Amino acid or nucleotide sequences were aligned with ClustalW Version 1.8. Construction of Expression Plasmids—The coding sequences for APC1, APC2, and APC3 and their C-terminal domains (CTDs) were amplified by PCR from human testis cDNA (APC1 and its CTD) and human brain cDNA (APC2 and APC3 and their CTDs). For amplification of APC1, APC2, and APC3, forward and reverse oligonucleotide primers corresponded to the extremities of their coding sequences (GenBank™/EBI accession numbers AJ619961, AJ619962 and AJ619963, respectively). For amplification of the CTDs, the forward primers corresponded to nucleotides 619–642, 915–934, and 626–646 of the APC1, APC2, and APC3 cDNAs, respectively. Each pair of forward and reverse primers had HindIII and BamHI or BamHI and HindIII restriction sites as linkers. The reverse primers with additional BamHI sites did not contain the stop codon. The amplified products carrying HindIII and BamHI were cloned into a modified pcDNA3 expression vector (19Chiesa A. Rapizzi E. Tosello V. Pinton P. de Virgilio M. Fogarty K.E. Rizzuto R. Biochem. J. 2001; 355: 1-12Crossref PubMed Scopus (127) Google Scholar) in-frame with the HA1-EGFP sequence (where HA1 is a nine-amino acid epitope derived from hemagglutinin, and EGFP represents the humanized version of the S65T green fluorescent protein (GFP) mutant). The amplified products carrying BamHI and HindIII were cloned into the E. coli pQE30 expression vector (QIAGEN Inc.). The cloning of APC3 and its CTD was preceded by a site-directed mutagenesis reaction to remove the BamHI site present in their coding sequence. The pcDNA3 and pQE30 constructs were transformed into E. coli DH5α cells. Transformants were selected on 2XTY (16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, pH 7.4) plates containing ampicillin (100 μg/ml) and screened by direct colony PCR. The sequences of inserts were verified. Bacterial Expression and Purification—The expression of the three APC proteins and of their CTDs was carried out at 37 °C in E. coli M15(pREP4) (QIAGEN Inc.) according to the manufacturer's instructions. Control cultures with the empty vector were processed in parallel. Inclusion bodies were purified on a sucrose density gradient (20Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (184) Google Scholar) and washed at 4 °C first with 10 mm Tris-HCl and 1 mm EDTA (pH 7.0); twice with 3% (w/v) Triton X-114, 1 mm EDTA, and 10 mm PIPES (pH 7.0); and finally with 10 mm PIPES (pH 7.0). The proteins were solubilized in 1.6% (w/v) Sarkosyl. Small residues were removed by centrifugation at 258,000 × g for 20 min at 4 °C. Reconstitution into Liposomes and Transport Measurements—The recombinant proteins in Sarkosyl were reconstituted into liposomes in the presence of cardiolipin (0.7 mg/ml for APC1 and its CTD and 0.3 mg/ml for APC2 CTD) and 20 mm PIPES-NaOH (pH 7.0) with or without substrate as described (21Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). External substrate was removed from proteoliposomes on Sephadex G-75 columns pre-equilibrated with 100 mm sucrose and 20 mm PIPES-NaOH (pH 7.0) (buffer A) or with 1 mm PIPES-NaOH (pH 7.0) in the experiments reported in Table III. Transport at 25 °C was started by addition of the indicated labeled substrate to substrate-loaded proteoliposomes (exchange) or to unloaded proteoliposomes (uniport). In both cases, transport was terminated by addition of 0.2% tannic acid (the “inhibitor-stop” method) (21Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). In controls, the inhibitor was added at the beginning together with the radioactive substrate. All transport measurements were carried out in the presence of 20 mm PIPES (pH 7.0) in the internal and external compartments, except in the experiments reported in Table III, where 1 mm PIPES (pH 7.0) was used. The external substrate was removed, and the radioactivity in the liposomes was measured (21Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). The experimental values were corrected by subtracting control values. The initial transport rate was calculated from the radioactivity taken up by proteoliposomes in the initial linear range of substrate uptake. For efflux measurements, proteoliposomes containing 2 mm substrate were labeled with 5 μm radioactive substrate by carrier-mediated exchange equilibration (21Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (229) Google Scholar). After 60 min, the external radioactivity was removed by passing the proteoliposomes through Sephadex G-75. Efflux was started by adding unlabeled external substrate or buffer A alone and terminated by adding the inhibitor indicated above.Table IIIInfluence of membrane potential and ΔpH on the activity of reconstituted APCs The exchanges were started by the addition of 0.3 mm [14C]ATP or 0.2 mm [14C]ATP-Mg to proteoliposomes containing various internal substrates (20 mm). 1 mm K+in was included as KCl in the reconstitution mixture, whereas 50 mm K+out was added as KCl together with the labeled substrates. 1 mm PIPES-NaOH (pH 7.0) was present in the internal and external compartments. Valinomycin or nigericin was added in 10 μl of ethanol/ml of proteoliposomes. In the control samples, the solvent alone was added. The exchange reactions were stopped after 30 s. Similar results were obtained in three independent experiments.Carrier and uptakeInternal substrateTransport activityControl+Valinomycin+Nigericinμmol/min/g proteinAPC1[14C]ATP-MgATP-Mg193178184[14C]ATP-MgATP14514995[14C]ATP-MgPi120107113[14C]ATP-MgNone1.31.21.0[14C]ATPATP-Mg200208297[14C]ATPATP154150147[14C]ATPPi107103176[14C]ATPNone1.71.51.8APC2 CTD[14C]ATP-MgATP-Mg373941[14C]ATP-MgATP343723[14C]ATP-MgPi323033[14C]ATP-MgNone0.50.80.5[14C]ATPATP-Mg424259[14C]ATPATP352934[14C]ATPPi282540[14C]ATPNone0.70.40.8 Open table in a new tab Expression Analysis by Real-time PCR—Total RNAs from human tissues (Invitrogen) were reverse-transcribed with the GeneAmp RNA PCR Core kit (Applied Biosystems) with random hexamers as primers. For real-time PCRs, primers and probes based on the cDNA sequences of APC1, APC2, and APC3 were designed with Primer Express (Applied Biosystems). The forward and reverse primers corresponded to nucleotides 143–169 and 226–252 (APC1), nucleotides 247–269 and 306–326 (APC2), and nucleotides 490–510 and 550–575 (APC3), respectively. The carboxyfluorescein-Dark Quencher-labeled probes corresponded to nucleotides 171–197, 272–293, and 513–537 of the APC1, APC2, and APC3 cDNAs, 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-strand cDNA), 1× TaqMan universal master mixture (Applied Biosystems), 200 nm probe (for APC1, APC2, or APC3), and 900 nm each primer. To correct for differences in the amount of starting first-strand cDNAs, the human β-actin gene was amplified in parallel as a reference gene. The relative quantification of APC1, APC2, or APC3 in various tissues was performed according to the comparative method (2-ΔΔCt) (22Bustin S.A. J. Mol. Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3103) Google Scholar, 23Fiermonte G. Palmieri L. Todisco S. Agrimi G. Palmieri F. Walker J.E. J. Biol. Chem. 2002; 277: 19289-19294Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar; User Bulletin 2 P/N 4303859, Applied Biosystems), with the pancreas ΔCt for APC1, the spleen ΔCt for APC2, and the liver ΔCt for APC3 as internal calibrators. 2-ΔΔCt = 2-(ΔCt sample-ΔCt calibrator), where ΔCt sample is Ct sample - Ct reference gene and Ct is the threshold cycle, i.e. the PCR cycle number at which emitted fluorescence exceeds 10 times the S.D. of base-line emissions. Subcellular Localization—For the subcellular localization of the three APC proteins and their CTDs in Chinese hamster ovary cells, the cells were grown on 24-mm round coverslips to 50–70% confluence and cotransfected according to a standard calcium phosphate procedure with 3 μg of pcDNA1/mtEBFP (24Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1856) Google Scholar); 3 μg of modified pcDNA3 plasmid containing the coding sequence of APC1, APC2, or APC3 or each of their CTDs fused with the EGFP sequence; and (in some experiments) 3 μg of DsRed2-peroxi plasmid (Clontech). About 30–40% of the cells were transfected. EGFP, enhanced blue fluorescent protein (BFP), and peroxisome-targeted DsRed2 fluorescence was detected by an inverted Zeiss Axiovert 200 microscope equipped with epifluorescence. Cells were imaged with a CoolSNAP HQ CCD camera (Roper Scientific, Trenton, NJ) using Metamorph software (Universal Imaging Corp., Downingtown, PA). Other Methods—A K+ diffusion potential was generated by adding valinomycin (1.5 μg/mg of phospholipid) to proteoliposomes in the presence of a potassium gradient. For the formation of ΔpH (acidic outside), nigericin (50 ng/mg of phospholipid) was added to proteoliposomes in the presence of an inwardly directed K+ gradient. Proteins were separated by SDS-PAGE and stained with Coomassie Blue. The N termini were sequenced, and the amount of purified proteins was estimated by laser densitometry of stained samples using carbonic anhydrase as a protein standard (25Fiermonte G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: 22782-22787Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The amount of protein incorporated into liposomes was measured as described (25Fiermonte G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: 22782-22787Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In all cases, it was ∼30% of the protein added to the reconstitution mixture. All of the specific activities reported in this study were calculated using the amount of inserted protein. Identification and Characterization of the APC1, APC2, and APC3 cDNAs—The sequence of human AAC1 (encoded by the SLC25A4 gene) was used to try to find candidates for human APC. Several related EST sequences were found in human EST data bases; we considered only the clones that encode proteins of unknown function and that contain Ca2+-binding EF-hand motifs in their sequences because it had been shown that ATP-Mg/Pi carrier activity is stimulated by calcium (13Haynes Jr., R.C. Picking R.A. Zaks W.J. J. Biol. Chem. 1986; 261: 16121-16125Abstract Full Text PDF PubMed Google Scholar, 14Nosek M.T. Dransfield D.T. Aprille J.R. J. Biol. Chem. 1990; 265: 8444-8450Abstract Full Text PDF PubMed Google Scholar, 17Dransfield D.T. Aprille J.R. Am. J. Physiol. 1993; 264: C663-C670Crossref PubMed Google Scholar). Three related human EST clones (GenBank™/EBI accession numbers AF123303, AK054901, and AB067483) matched these criteria; they were extended to the 3′- and 5′-ends, and their full-length cDNAs were obtained and sequenced. The final cDNAs (GenBank™/EBI accession numbers AJ619961 (for APC1), AJ619962 (for APC2), and AJ619963 (for APC3)) consisted of 3345 (APC1), 3430 (APC2), and 3660 (APC3) nucleotides and encoded protein sequences of 458, 468, and 489 amino acids, respectively. These proteins belong to a subfamily of Ca2+-binding mitochondrial carriers with a characteristic bipartite structure. Their N-terminal domains (the first 166, 176, and 196 amino acids of APC1, APC2, and APC3, respectively) contain three EF-hand Ca2+-binding motifs, as shown in Fig. 1, and have 43–46% identical amino acids. The three C-terminal domains of the APCs contain the common sequence features of the mitochondrial carrier family, and their amino acids are 66–75% identical to each other and 27–30% identical to those of human AAC1. They also contain a sequence at residues 319–323 (APC1), 329–333 (APC2), and 350–354 (APC3) in the second matrix loop that conforms to the sequence motif EGXXA, the P-box of the DNA-binding domain of nuclear receptors (26Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (607) Google Scholar). The same motif is present in the deoxynucleotide carrier, the ADP/ATP carrier, and the uncoupling protein UCP1 (27Dolce V. Fiermonte G. Runswick M.J. Palmieri F. Walker J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2284-2288Crossref PubMed Scopus (165) Google Scholar, 28Muller V. Basset G. Nelson D.R. Klingenberg M. Biochemistry. 1996; 35: 16132-16143Crossref PubMed Scopus (59) Google Scholar, 29Gonzalez-Barroso M.M. Fleury C. Jimenez M.A. Sanz J.M. Romero A. Bouillaud F. Rial E. J. Mol. Biol. 1999; 292: 137-149Crossref PubMed Scopus (27) Google Scholar) and may be involved in the binding of nucleotide substrates because it was shown to bind purine nucleotides in rat UCP1 (29Gonzalez-Barroso M.M. Fleury C. Jimenez M.A. Sanz J.M. Romero A. Bouillaud F. Rial E. J. Mol. Biol. 1999; 292: 137-149Crossref PubMed Scopus (27) Google Scholar). By screening the human genome data bases with the three APC cDNAs, the corresponding genes were found. The APC1 gene (gene name SLC25A24) is spread over 58 kb on chromosome 1p13.3; the APC2 gene (gene name SLC25A23) is spread over 20 kb on chromosome 19p13.3; and the APC3 gene (gene name SLC25A25) is spread over 18 kb on chromosome 9q34.13. All three genes contain 10 exons separated by nine introns. In the three genes, all of the splicing junctions occur in the same nucleotide regions, indicating a triplication of a common ancestral gene. Expression of Human APC1, APC2, and APC3 in Various Tissues—The tissue distribution of mRNAs for the human APC proteins determined by real-time PCR is summarized in Fig. 2. The amount of APC1 mRNA in the pancreas was the same as the amount of APC3 mRNA in the liver (as they have the same ΔCt in these tissues), and so this value served as an internal calibration in the relative quantification of these two proteins in various tissues. In all of the tissues investigated except testis, the APC3 mRNA was expressed at higher levels than the APC1 mRNA. It was expressed most strongly in the brain, testis, and lung; in reasonable abundance in the small intestine, pancreas, skeletal muscle, and heart; and at lower levels in the kidney, spleen, and liver. The APC1 mRNA was expressed in very abundant amounts in the testis; at low levels in the small intestine and pancreas; and poorly in the kidney, spleen, liver, skeletal muscle, and heart. It was not detectable in the brain and lung. For APC2, the ΔCt in the spleen was used as an internal calibrator in its relative quantification in various tissues. APC2 was expressed in the kidney, lung, small intestine, pancreas, liver, and heart and especially in the brain and skeletal muscle at levels severalfold higher than the amount present in the spleen. Only in the testis was the APC2 mRNA expressed at a level comparable with that in the spleen. Although the levels of expression of APC2 (Fig. 2) were not directly comparable with those of APC1 and APC3, it is noteworthy that the ΔCt of APC2 mRNA in the spleen was lower than that used as an internal calibrator for APC1 and APC3; and consequently, the amount of APC2 mRNA in the spleen was higher than that of APC1 and APC3 in the tissues used as internal calibrators (pancreas and liver, respectively). It should also be borne in mind that, as post-transcriptional mechanisms may be in operation, the levels of mRNAs reported in Fig. 2 do not necessarily reflect the ratios of transport activities. Bacterial Expression of APC Proteins—APC1, APC3, and the CTDs of APC1 and APC2 (corresponding to the last 292 amino acids of APC1 and APC2) were overexpressed in E. coli M15(pREP4) (Fig. 3, see lanes 2, 4, and 6 for APC1 CTD, APC2 CTD, and APC1, respectively). They accumulated as inclusion bodies and were purified by centrifugation and washing. The apparent molecular masses of the purified proteins (yield of 60–80 mg/liter) (Fig. 3, lanes 7–10) were ∼51 and 32 kDa for APC1 and its CTD, respectively; 32.5 kDa for APC2 CTD; and 55 kDa for APC3, in good agreement with the calculated values. Their identities were confirmed by N-terminal sequencing. The proteins were not detected in bacteria harvested immediately before the induction of expression (Fig. 3, lanes 1, 3, and 5) or in cells harvested after induction, but lacking the coding sequence in the expression vector (data not shown). APC2 and APC3 CTD were not expressed at appreciable levels in E. coli M15(pREP4) or in E. coli BL21(DE3) or CO214(DE3) using the pET expression system. Functional Characterization of Recombinant APC Proteins— All four recombinant proteins were reconstituted into liposomes, and their transport activities for a large variety of potential substrates were tested in homo-exchange experiments (i.e. with the same substrate inside and outside). Using external and internal substrate concentrations of 1 and 20 mm, respectively, reconstituted APC1, APC1 CTD, and APC2 CTD catalyzed active [14C]ADP/ADP and [33P]Pi/Pi exchanges, which were totally inhibited by 0.2% tannic acid. They did not catalyze homo-exchanges for pyruvate, succinate, malate, oxoglutarate, ketoisocaproate, citrate, carnitine, ornithine, lysine, arginine, glutamate, aspartate, histidine, glutathione, choline, spermine, proline, and threonine. Of importance, no [14C]ADP/ADP or [33P]Pi/Pi exchange activity was detected if APC1, APC1 CTD, and APC2 CTD had been boiled before incorporation into liposomes or if proteoliposomes were reconstituted with Sarkosyl-solubilized material from bacterial cells lacking the expression vector for APC1, APC1 CTD, or APC2 CTD or harvested immediately before induction of expression. At variance with APC1, APC1 CTD, and APC2 CTD, recombinant and reconstituted APC3 showed no activity under any of the experimental conditions tested, which included variation of the parameters that influence solubilization of the inclusion bodies and reconstitution of the protein into liposomes. The substrate specificities of reconstituted APC1, APC1 CTD, and APC2 CTD were examined in detail by measuring the uptake of [14C]ADP or [33P]Pi into proteoliposomes that had been preloaded with various potential substrates (Table I). With APC1 and its CTD, the highest activities were observed in the presence of internal ATP-Mg, ADP, Pi, AMP, and ATP. Interestingly, with ATP (but not ADP), the uptake of labeled substrate was greater in the presence of internal Mg2+ than in its absence. [14C]ADP and [33P]Pi also exchanged with internal deoxynucleotides of adenine, 3′-AMP, 3′,5′-ADP, and pyrophosphate, although to a lesser extent. A low activity was observed with G, C, T, and U nucleotides. In contrast, the uptake of labeled substrates was negligible in the presence of internal NaCl and cAMP, adenosine, thiamine, thiamine mono- and diphosphates, FAD, NAD, NMN, S-adenosylmethionine, 5-phospho-α-d-ribosyl-1-pyrophosphate, succinate, fumarate, aspartate, glutamate, malate, malonate, citrate, oxoglutarate, oxalacetate, pyruvate, phosphoenolpyruvate, l-carnitine, l-ornithine, and l-citrulline (data not shown). Reconstituted APC2 CTD behaved similarly to APC1 and its CTD (Table I). However, the rates of the APC2 CTD-mediated [14C]ADP and [33P]Pi exchanges were lower than the corresponding rates catalyzed by APC1 and its CTD.Table IDependence on internal substrate of the transport properties of proteoliposomes reconstituted with recombinant APC1, APC1 CTD, and APC2 CTD Proteoliposomes were preloaded internally with various substrates (20 mm). Transport was started by the addition of 0.3 mm [14C]ADP or 1.5 mm [33P]Pi to proteoliposomes reconstituted with APC1 and APC1 CTD or by the addition of 0.5 mm [14C]ADP or 1.5 mm [33P]Pi
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