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• W1983411754 abstract "Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1–82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling. Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1–82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling. Carnitine palmitoyltransferase 1 (CPT) 3The abbreviations used are:CPT1carnitine palmitoyltransferase 1CPT1Aliver isoform of CPT1CPT1Bmuscle isoform of CPT1CACTcarnitine/acylcarnitine translocaseLCFAlong chain fatty acidOMMouter mitochondrial membraneIMMinner mitochondrial membraneTMtransmembrane segmentIMSintermembrane spaceDHFRdihydrofolate reductasesulfo-GMBSN-(γ-maleimidobutyloxy)-sulfosuccinimide estersulfo-MBSm-maleimidobenzoyl-N-hydroxysulfosuccinimide estersulfo-KMUSN-(κ-maleimidoundecanoyloxy)-sulfosuccinimide esterDSSdisuccinimidyl suberateBN-PAGEblue native gel electrophoresisCOX IVsubunit IV of the cytochrome c oxidasecyt b2cytochrome b2COTcarnitine octanoyltransferaseCrATcarnitine acetyltransferaseBistris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (EC 2.3.1.21) is the key regulatory enzyme of mitochondrial long chain fatty acid (LCFA) oxidation (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1354) Google Scholar, 2Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (311) Google Scholar). This enzyme catalyzes the conversion of long chain acyl-CoA to acylcarnitines, which permits, in cooperation with the carnitine/acylcarnitine translocase (CACT) and the CPT2, their transport from the cytosol into the mitochondrial matrix to undergo β-oxidation. CPT1 is an integral protein of the outer mitochondrial membrane (OMM) and exists under two main isoforms, the liver (CPT1A) and the muscle (CPT1B), whereas CPT2 is loosely associated with the inner face of the inner mitochondrial membrane (IMM), and only one ubiquitous isoform exists (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1354) Google Scholar). A unique feature of CPT1 is its potent inhibition by malonyl-CoA (3McGarry J.D. Leatherman G.F. Foster D.W. J. Biol. Chem. 1978; 253: 4128-4136Abstract Full Text PDF PubMed Google Scholar), the first committed intermediate in fatty acid biosynthesis. This malonyl-CoA/CPT1 partnership is a key actor not only in physiological regulation of LCFA oxidation (1McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1354) Google Scholar) but also in many other processes such as fuel-sensing in hypothalamic neurons and the regulation of food intake and energy homeostasis (4Wolfgang M.J. Lane M.D. J. Biol. Chem. 2006; 281: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). carnitine palmitoyltransferase 1 liver isoform of CPT1 muscle isoform of CPT1 carnitine/acylcarnitine translocase long chain fatty acid outer mitochondrial membrane inner mitochondrial membrane transmembrane segment intermembrane space dihydrofolate reductase N-(γ-maleimidobutyloxy)-sulfosuccinimide ester m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester N-(κ-maleimidoundecanoyloxy)-sulfosuccinimide ester disuccinimidyl suberate blue native gel electrophoresis subunit IV of the cytochrome c oxidase cytochrome b2 carnitine octanoyltransferase carnitine acetyltransferase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Rat CPT1A (88 kDa) and CPT2 (68 kDa) share 50% homology in the major part of their sequences with the exception of their N termini (5Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. J. Biol. Chem. 1993; 268: 5817-5822Abstract Full Text PDF PubMed Google Scholar). The extended N-terminal domain (about 150 residues) of CPT1A bears no significant similarity to CPT2 and contains two hydrophobic transmembrane (TM1 and TM2) segments (5Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. J. Biol. Chem. 1993; 268: 5817-5822Abstract Full Text PDF PubMed Google Scholar). Its N terminus (residues 1–47) and its large catalytic C-terminal domain (residues 123–773) are exposed on the cytosolic face of mitochondria, whereas the loop connecting TM1 and TM2 protrudes into the intermembrane space (IMS) (6Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (124) Google Scholar). The N-terminal domain is essential for mitochondrial targeting, for import into the OMM, and for maintenance of a folded active and malonyl-CoA-sensitive conformation (7Cohen I. Kohl C. McGarry J.D. Girard J. Prip-Buus C. J. Biol. Chem. 1998; 273: 29896-29904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Pioneer studies showed that inhibition by malonyl-CoA is produced by the occurrence of two binding sites. One site is the low affinity site corresponding to the catalytic acyl-CoA binding domain leading to a competition behavior between malonyl-CoA and palmitoyl-CoA, whereas the second high affinity site is distinct from the active site and does not compete with acyl-CoA (3McGarry J.D. Leatherman G.F. Foster D.W. J. Biol. Chem. 1978; 253: 4128-4136Abstract Full Text PDF PubMed Google Scholar, 9Bird M.I. Saggerson E.D. Biochem. J. 1984; 222: 639-647Crossref PubMed Scopus (42) Google Scholar, 10Grantham B.D. Zammit V.A. Biochem. J. 1986; 239: 485-488Crossref PubMed Scopus (48) Google Scholar). Functional studies indicated that both malonyl-CoA-binding sites of CPT1A are located within its catalytic C-terminal domain (11Morillas M. Gomez-Puertas P. Rubi B. Clotet J. Arino J. Valencia A. Hegardt F.G. Serra D. Asins G. J. Biol. Chem. 2002; 277: 11473-11480Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and not within its cytosolic N terminus (12Pan Y. Cohen I. Guillerault F. Feve B. Girard J. Prip-Buus C. J. Biol. Chem. 2002; 277: 47184-47189Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). However, the latter is essential for maintaining the high affinity malonyl-CoA-binding site (13Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). We have demonstrated that the N terminus of CPT1A, which contains both positive and negative determinants of malonyl-CoA sensitivity (14Jackson V.N. Zammit V.A. Price N.T. J. Biol. Chem. 2000; 275: 38410-38416Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Jackson V.N. Price N.T. Zammit V.A. Biochemistry. 2001; 40: 14629-14634Crossref PubMed Scopus (24) Google Scholar), influences the degree of malonyl-CoA sensitivity through modulations of its interaction with the C-terminal domain (16Faye A. Borthwick K. Esnous C. Price N.T. Gobin S. Jackson V.N. Zammit V.A. Girard J. Prip-Buus C. Biochem. J. 2005; 387: 67-76Crossref PubMed Scopus (33) Google Scholar). Moreover, the existence of the two malonyl-CoA-binding sites, as well as the importance of these intramolecular N/C interactions for malonyl-CoA sensitivity, was recently supported by a three-dimensional in silico model of both the N- and C-terminal domains (17Lopez-Vinas E. Bentebibel A. Gurunathan C. Morillas M. de Arriaga D. Serra D. Asins G. Hegardt F.G. Gomez-Puertas P. J. Biol. Chem. 2007; 282: 18212-18224Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Although key insights into the structure-function relationships of CPT1A have emerged during the past decade, the quaternary structure of CPT1A still remains totally unknown. Previously reported hysteretic behavior and intrinsic positive cooperativity of CPT1A for palmitoyl-CoA (3McGarry J.D. Leatherman G.F. Foster D.W. J. Biol. Chem. 1978; 253: 4128-4136Abstract Full Text PDF PubMed Google Scholar, 18Saggerson E.D. Carpenter C.A. FEBS Lett. 1981; 132: 166-168Crossref PubMed Scopus (40) Google Scholar, 19Cook G.A. J. Biol. Chem. 1984; 259: 12030-12033Abstract Full Text PDF PubMed Google Scholar, 20Pauly D.F. McMillin J.B. J. Biol. Chem. 1988; 263: 18160-18167Abstract Full Text PDF PubMed Google Scholar) suggested multisubunit cooperation. Moreover, the observation of positive cooperative inhibition of CPT1A by malonyl-CoA and that malonyl-CoA could introduce sigmoidicity into the relationship between palmitoyl-CoA and CPT1A activity (3McGarry J.D. Leatherman G.F. Foster D.W. J. Biol. Chem. 1978; 253: 4128-4136Abstract Full Text PDF PubMed Google Scholar, 18Saggerson E.D. Carpenter C.A. FEBS Lett. 1981; 132: 166-168Crossref PubMed Scopus (40) Google Scholar, 21Cook G.A. J. Biol. Chem. 1987; 262: 4968-4972Abstract Full Text PDF PubMed Google Scholar) strongly suggested allosteric behavior. Binding of malonyl-CoA could produce a conformational change that would either make the binding of a second malonyl-CoA much easier or make the binding of a second long chain acyl-CoA more difficult. Proteins exhibiting such cooperative effects are thought to be complexes of two or more subunits, raising the crucial question of whether CPT1A exists as a monomer or is assembled into an oligomeric complex once imported into the OMM. In this study we have used three complementary approaches, namely chemical cross-linking, chromatography by gel filtration, and blue native PAGE, to study for the first time the quaternary structure of rat CPT1A. We show evidence that both CPT1A expressed in Saccharomyces cerevisiae and native protein from fed rat liver mitochondria self-assemble into an oligomeric complex and that the fundamental unit of their quaternary structure is a homotrimer. Moreover, the oligomeric state of CPT1A was unchanged by starvation and diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, by using several fusions or partially deleted CPT constructs, we demonstrated that the N-terminal domain of CPT1A can trigger its oligomerization, but the latter is limited to a trimer likely by the presence of its large catalytic C-terminal domain on the cytosolic face of the mitochondria. The functional relevance of the existence of an oligomeric CPT1A complex within the OMM is discussed. Yeast Expression of Fusion and Deletion Proteins—The S. cerevisiae strains (haploid strain W303: MATa, his3, leu2, trp1, ura3, ade2-1, can1-100) expressing either rat CPT1A, CPT1Δ1–82, pOM29-CPT2, or CPT1-(1–147)-CPT2 were obtained as described previously (7Cohen I. Kohl C. McGarry J.D. Girard J. Prip-Buus C. J. Biol. Chem. 1998; 273: 29896-29904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Briefly, pOM29-CPT2 corresponds to the fusion of the first N-terminal 29 amino acids of S. cerevisiae Tom70p, pOM29, which contains a specific OMM signal anchor sequence (23Millar D.G. Shore G.C. J. Biol. Chem. 1993; 268: 18403-18406Abstract Full Text PDF PubMed Google Scholar) to the mature form of CPT2. CPT1-(1–147)-CPT2 is the fusion of the first 147 N-terminal residues of CPT1A to the mature form of CPT2. CPT1Δ1–82 corresponds to CPT1A deleted of residues 1–82. For yeast expression of the dihydrofolate reductase (DHFR) fusion constructs, the corresponding cDNAs were retrieved from pGEM4 as an EcoRI-HindIII-blunted fragment (7Cohen I. Kohl C. McGarry J.D. Girard J. Prip-Buus C. J. Biol. Chem. 1998; 273: 29896-29904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and subcloned into the yeast expression vector pYeDP1–8/10 cut by EcoRI and SmaI (22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). These DHFR fusion constructs were CPT1-(1–147)-DHFR and CPT1-(97–147)-DHFR, which respectively correspond to the fusion of amino acids 1–147 and 97–147 of CPT1A to DHFR. A C-terminally Myc/His-6 tagged construct had been previously generated for expression in Pichia pastoris as follows. The PCR primers 5′-GCAGGTGGAGCTCTTTGACTTT-3′ and 5′-TTTTGGGCCCCTTTTTAGAATTGATGGTGAG-3′ were used to amplify a region corresponding to the C terminus of CPT1A, with replacement of the stop codon with an ApaI restriction site. The product was cleaved with SacI-ApaI and used to replace the 3′ end of CPT1A previously subcloned into pGAPZ A (Invitrogen). To generate the construct for S. cerevisiae expression, the SacI-BamHI fragment (including the region encoding the tag) was subcloned into empty pYeDP1–8/10. The remainder of CPT1A was then added as an EcoRI-SacI fragment. Each cDNA was placed under the control of the inducible GAL10 promoter present in the vector, and the constructs were used to transform S. cerevisiae. The fidelity of PCR and the quality of DNA subcloning were confirmed by DNA sequencing. Isolation of Yeast and Rat Liver Mitochondria—Methods for yeast culture and isolation of yeast mitochondria were performed as described previously (22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Rat liver mitochondria were isolated from male Wistar rats (200–300 g) (Centre d'Elevage et de Reproduction Janvier, Le Genest St. Isle, France) that had continuous free access to water and were either fed ad libitum on a standard laboratory chow diet (62% carbohydrate, 12% fat, and 26% protein in terms of energy) or starved for 48 h. Diabetes was induced by a single intraperitoneal injection of streptozotocin as described previously (16Faye A. Borthwick K. Esnous C. Price N.T. Gobin S. Jackson V.N. Zammit V.A. Girard J. Prip-Buus C. Biochem. J. 2005; 387: 67-76Crossref PubMed Scopus (33) Google Scholar). All animals were kept on a light/dark cycle (light from 15:00 to 03:00 h) and were killed at 08:00 h. Rat liver mitochondria were isolated in an isolation buffer (0.3 m sucrose, 5 mm Tris-HCl, 1 mm EGTA, pH 7.4) using differential centrifugation, further purified on self-forming Percoll gradients, and resuspended in the isolation buffer as described previously (7Cohen I. Kohl C. McGarry J.D. Girard J. Prip-Buus C. J. Biol. Chem. 1998; 273: 29896-29904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Protein concentration was determined by the method of Lowry with bovine serum albumin as a standard. CPT1 Activity—CPT1 activity was assayed using freshly isolated rat liver mitochondria (0.1 mg of protein/ml) at 30 °C as palmitoyl-l-[methyl-3H]carnitine formed from l-[methyl-3H]carnitine (200 μm; 10 Ci/mol) and palmitoyl-CoA (80 μm) in the presence of 1% (w/v) bovine serum albumin as described previously (22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Malonyl-CoA concentration was varied over 0.01–150 μm for estimation of the IC50 value (concentration of malonyl-CoA required to achieve 50% inhibition at 80 μm palmitoyl-CoA). Chemical Cross-linking—Mitochondria were washed twice either in HSY buffer (10 mm Hepes, 0.6 m sorbitol, pH 8) (yeast mitochondria) or in HSR buffer (10 mm Hepes, 0.3 m sucrose, pH 8) (rat liver mitochondria), and finally resuspended in the respective buffer at a protein concentration of 2 mg/ml. The polar cross-linkers, sulfo-GMBS, sulfo-MBS, and sulfo-KMUS, used were dissolved in water. The apolar cross-linker, DSS, was dissolved in Me2SO. After addition of cross-linkers to a final concentration of either 50, 100, 200, 250, or 500 μm to mitochondria (80 μg), samples were incubated for 30 min at 4 °C. Excess of cross-linkers was then quenched by addition of 6 μlof TSY buffer (750 mm Tris-HCl, 0.6 m sorbitol, pH 8) (yeast mitochondria) or TSR buffer (750 mm Tris-HCl, 0.3 m sucrose, pH 8) (rat liver mitochondria). After incubation for 15 min at 4 °C, mitochondria were recovered by centrifugation, resuspended in 0.1 m Na2CO3, pH 11.5, at a final protein concentration of 0.2 mg/ml, and incubated on ice for 30 min. After centrifugation at 177,000 × g for 30 min at 4 °C, integral membrane proteins were recovered in the pellet, analyzed by SDS-PAGE in a 4–8% gradient gel, and immunoblotted. Detergent Solubilization of CPT1A—Mitochondria were reisolated by centrifugation for 10 min at 12,000 × g and resuspended at a protein concentration of 10 mg/ml either in the BN-PAGE buffer (750 mm 6-aminocaproic acid, 50 mm Bistris/HCl, 0.5 mm phenylmethylsulfonyl fluoride, 10% (v/v) glycerol, pH 7) containing either 1% (v/v) Triton X-100, 2 or 1% (w/v) digitonin, or 1% (v/v) SDS, or in the gel filtration buffer (1% (v/v) Triton X-100, 150 mm potassium acetate, 4 mm magnesium acetate, 30 mm Tris-HCl, 0.5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, pH 7.4). The lysis buffers were supplemented with 5 mg/ml aprotinin, 2 mg/ml leupeptin, and 1 mg/ml pepstatin. After solubilization for 20 min on ice, mitochondrial extracts were centrifuged for 30 min at 177,000 × g, and the supernatants were recovered. Gel Filtration Analysis—Supernatants from solubilized mitochondria (1 mg of protein) were loaded into either a Superose 6 or Superose 12 gel filtration column (25-ml column volume; Amersham Biosciences) and chromatographed in the gel filtration buffer at a flow rate of 0.2 ml/min. Fractions (0.2 ml) were collected, trichloroacetic acid-precipitated, analyzed by SDS-PAGE in a 8 or 12% gel, and immunoblotted. Calibration standards (Amersham Biosciences) used were as follows: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and albumin (67 kDa) for the Superose 6 column, and catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) for the Superose 12 column. BN-PAGE—BN-PAGE was performed as described previously (24Schagger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar). Briefly, following solubilization of mitochondria in the BN-PAGE buffer, supernatants (28.5 μl) were supplemented with 1.5 μl of sample buffer (5% (w/v) Serva Blue G in 500 mm 6-aminocaproic acid) prior to electrophoresis. Samples were then analyzed on a 4–13% gradient BN gel. The calibration standards used were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and albumin (67 kDa). Western Blot Analysis—Detection of proteins after blotting onto nitrocellulose was performed as described previously (22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar) using the ECL detection system (Pierce). The antibodies used were against the rat CPT1A (22Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar), the mouse DHFR (BD Biosciences), the His tag (Novagen), the yeast cytochrome b2 (gift from Prof. W. Neupert, Munich, Germany), and the rat Tom40 (gift from Prof. K. Mihara, Japan). For the generation of anti-CPT2 polyclonal antibody, peptide corresponding to the last 20 C-terminal residues of human CPT2 was synthesized, conjugated to keyhole limpet hemocyanin, and used to immunized New Zealand White rabbits (Neosystem, Strasbourg, France). The immunoblots were quantified using a Chemigenius apparatus (Syngene). Chemicals—PCR reagents, restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs (Ozyme, Saint-Quentin en Yvelines, France). Yeast culture media products were from Difco, and Zymolase 20T was from ICN Biomedicals, France. All cross-linkers were purchased from Pierce. Other chemicals were purchased from Sigma. CPT1A Exists as an Oligomeric Complex within the OMM—The first approach used to investigate the organization of rat CPT1A within the OMM was chemical cross-linking using intact mitochondria isolated from fed adult rat liver. After treatment with the noncleavable water-insoluble DSS (11.3 Å) cross-linker, CPT1A-containing cross-linking products were analyzed by SDS-PAGE and immunoblotting. Whereas the monomeric form of CPT1A (88 kDa) decreased as the concentration of DSS increased, both a major band corresponding to an apparent molecular mass ranging between 250 and 400 kDa and a minor band corresponding to an apparent molecular mass of ∼100 kDa were detected (Fig. 1A). Treatment with the noncleavable water-soluble sulfo-GMBS (6.8 Å), sulfo-MBS (9 Å), and sulfo-KMUS (15.7 Å) cross-linkers also led to the generation of these two specific CPT1A adducts. In addition, another band with an apparent molecular mass of ∼160 kDa was detected upon cross-linking with sulfo-GMBS and sulfo-MBS, which have a shorter spacer-arm length (Fig. 1A). These results suggested that within intact OMM of rat liver mitochondria, CPT1A interacts with protein(s) and may be assembled into complex(es). To determine whether the high molecular cross-linked product could result from oligomerization of CPT1A or not, cross-linking experiments were performed using mitochondria isolated from S. cerevisiae expressing rat CPT1A. Indeed, the yeast model is not only devoid of any of the mitochondrial CPTs but also of all the mitochondrial enzymes necessary for mitochondrial LCFA oxidation because this pathway takes place in peroxisomes. Therefore, the use of the yeast heterologous expression model specifically allowed us to study the oligomeric state of rat CPT1A when expressed in this model. As shown in Fig. 1B, treatment of mitochondria isolated from yeast cells expressing rat CPT1A with the sulfo-GMBS, sulfo-MBS, and sulfo-KMUS cross-linkers resulted in the generation of a high molecular mass band (∼200–360 kDa), whereas the ∼100-kDa band could never be detected. These observations suggested that, independently of the existence of a mitochondrial LCFA oxidation “metabolon” or of other protein-protein interactions, CPT1A could form an oligomeric complex within the OMM. Gel Filtration Chromatography Analysis of CPT1A Complex(es)—To demonstrate the existence of CPT1A complex(es), Superose 6 gel filtration chromatography experiments were performed after lysis of purified rat liver mitochondria with 1% (v/v) Triton X-100 under a low detergent to protein ratio. Internal protein control was the IMM COX IV (17 kDa), which is 1 of the 13 subunits of the cytochrome c oxidase (180 kDa) that exists as a dimer of monomers (25Suarez M.D. Revzin A. Narlock R. Kempner E.S. Thompson D.A. Ferguson-Miller S. J. Biol. Chem. 1984; 259: 13791-13799Abstract Full Text PDF PubMed Google Scholar). Western blot analysis of the eluted fractions indicated that rat liver COX IV was eluted as a single peak (Fig. 2A) corresponding to a molecular mass of ∼400 kDa (Fig. 2B). These observations validated that our experimental conditions did not dissociate this well known mitochondrial complex. The native rat CPT1A was eluted as a single peak (Fig. 2A), and the corresponding molecular mass of the detected complex was ∼275 kDa (Fig. 2B). We next addressed whether this complex may represent an oligomeric and/or multiproteic CPT1A complex by performing gel filtration experiments using solubilized mitochondrial extracts from yeast cells expressing rat CPT1A. Internal protein control for these experiments was the yeast cytochrome b2 (cyt b2) protein (52 kDa) that is assembled into a 250-kDa complex within the IMM (26Arlt H. Tauer R. Feldmann H. Neupert W. Langer T. Cell. 1996; 85: 875-885Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). As expected, Western blot analysis of the eluted fractions indicated that yeast cyt b2 was eluted as a single peak (Fig. 2A) corresponding to a molecular mass of ∼260 kDa (Fig. 2B). As observed for the native protein, the yeast-expressed CPT1A was also fractionated as an ∼275-kDa complex (Fig. 2, A and B). Similar results were obtained when rat liver or yeast mitochondria were solubilized with 1% (w/v) digitonin (results not shown). Because CPT1A has a molecular mass of 88 kDa, the results obtained using the yeast-expressed CPT1A revealed that CPT1A forms a homotrimeric complex within the OMM. In the native environment, i.e. rat liver OMM, the size of the ∼275-kDa CPT1A species (Fig. 2A) indicated that this trimeric organization is also preserved following solubilization and gel filtration chromatography, whereas other protein-protein interactions, as suggested by our cross-linking experiments (Fig. 1A), were likely disrupted under this experimental procedure. Native Gel Electrophoresis Analysis of CPT1A Complex(es)—To further examine the complex state(s) of CPT1A, detergent-solubilized rat liver mitochondria were subjected to BN-PAGE followed by Western blotting. Whatever the detergent (Triton X-100 or digitonin) used, the major fraction of CPT1A migrated as an ∼550–620-kDa complex (Fig. 3A). In addition, bands with a much lower intensity were observed and estimated at ∼130–150 and ∼90 kDa, the latter most likely corresponding to the monomeric form of CPT1A. CPT1A complexes solubilized with Triton X-100 were observed to migrate faster in BN-PAGE than those solubilized with digitonin (Fig. 3A). Such a difference between the two detergents could be explained by variation in the lipid content of the various complexes as reported previously (27Dembowski M. Kunkele K.P. Nargang F.E. Neupert W. Rapaport D. J. Biol. Chem. 2001; 276: 17679-17685Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Upon solubilization of rat liver mitochondria with the denaturing SDS detergent, only the ∼90-kDa signal could be detected but with a much higher intensity (Fig. 3A), indicating SDS-induced dissociation of CPT1A complexes. To validate our results, Tom40 (40 kDa) was used as an internal control because this membrane protein, which belongs to the OMM protein import machinery, is assembled into a ∼400-kDa complex (28Suzuki H. Okazawa Y. Komiya T. Saeki K. Mekada E. Kitada S. Ito A. Mihara K. J. Biol. Chem. 2000; 275: 37930-37936Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). As shown in Fig. 3A, Tom40 migrated at a molecular mass of ∼400 kDa indicating that our experimental conditions allowed solubilization of mitochondrial complexes without their disruption. We next addressed whether the ∼550–620-kDa species may represent an oligomeric" @default.
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