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• W1984418909 abstract "The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1–150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain ofL-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity. The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1–150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain ofL-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity. carnitine palmitoyltransferase liver CPT outer and inner mitochondrial membrane, respectively polymerase chain reaction dihydrofolate reductase soybean trypsin inhibitor polyacrylamide gel electrophoresis carbonyl cyanidem-chlorophenylhydrazone mitochondrial membrane potential. The mitochondrial carnitine palmitoyltransferase (CPT1; EC 2.3.1.21) system in cooperation with the carnitine/acylcarnitine translocase permits long-chain acyl-CoA to be transferred from the cytosol to the mitochondrial matrix to undergo β-oxidation (1McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (292) Google Scholar). The CPT system consists of two membrane-bound enzymes, CPT1 and CPT2. CPT1 is an integral protein of the outer mitochondrial membrane (OMM), loses activity upon solubilization of mitochondria by strong detergents and exists under two isoforms, the liver (L-CPT1; 88 kDa) and the muscle (82 kDa) (2McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1354) Google Scholar). By contrast, CPT2 is loosely associated with the inner face of the inner mitochondrial membrane (IMM), is released in active soluble form by detergents, and only one ubiquitous isoform exists (2McGarry 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, the first committed intermediate of fatty acid biosynthesis (3McGarry J.D. Mannaerts G.P. Foster D.W. J. Clin. Invest. 1977; 60: 265-270Crossref PubMed Scopus (519) Google Scholar). This provides a mechanism for physiological regulation of β-oxidation in liver and other tissues (1McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (292) Google Scholar, 4Girard J. Ferré P. Pégorier J.P. Duée P.H. Physiol. Rev. 1992; 72: 507-562Crossref PubMed Scopus (419) Google Scholar). Apart from mitochondria, rat liver microsomes and peroxisomes contain also both membrane-bound/malonyl-CoA-sensitive and soluble/malonyl-CoA-insensitive (luminal) CPT-like enzymes (5Derrick J. Ramsay R. Biochem. J. 1989; 262: 801-806Crossref PubMed Scopus (61) Google Scholar, 6Pande S.V. Bhuiyan A.K.M.J. Murthy M.S.R. Carter A.L. Current Concepts in Carnitine Research. 1992: 165-178Google Scholar, 7Murthy M.S.R. Pande S.V. J. Biol. Chem. 1994; 269: 18283-18286Abstract Full Text PDF PubMed Google Scholar, 8Broadway N.M. Saggerson E.D. Biochem. J. 1995; 310: 989-995Crossref PubMed Scopus (29) Google Scholar), which share similar functional properties with their mitochondrial counterparts. Thus, a similar fatty acid transport operates in mitochondria, microsomes, and peroxisomes, but it seems that the components involved in these systems are all different (6Pande S.V. Bhuiyan A.K.M.J. Murthy M.S.R. Carter A.L. Current Concepts in Carnitine Research. 1992: 165-178Google Scholar). This raised the crucial question of how L-CPT1 is specifically targeted to mitochondria and inserted into the OMM. Most mitochondrial proteins are encoded by nuclear genes, synthesized as precursors in the cytosol, targeted to mitochondria, and finally imported into their respective mitochondrial subcompartments. Mitochondrial targeting and transport is due to the interaction of targeting signals within the precursors with an import receptor complex located in the OMM and named the Tom complex (9Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar). In the case of most matrix proteins, and especially for CPT2 (10Brown N.F. Esser V. Gonzalez A.D. Evans C.T. Slaughter C.A. Foster D.W. McGarry J.D. J. Biol. Chem. 1991; 266: 15446-15449Abstract Full Text PDF PubMed Google Scholar), their N-terminal presequences function as cleavable matrix-targeting signals that initiate translocation across both mitochondrial membranes in a membrane potential-dependent fashion. By contrast, OMM proteins do not contain cleavable signal presequences and therefore must be targeted to mitochondria by means of internal signals. How this is accomplished is still not clear, although clues have begun to emerge from studies of bitopic proteins, such as the yeast Tom70p and the human Bcl2 (11Shore G.M., H. Millar D. Steenaart N. Nguyen M. Eur. J. Biochem. 1995; 227: 9-18Crossref PubMed Scopus (84) Google Scholar). Their targeting and insertion into the OMM have been shown to be mediated by a unique hydrophobic transmembrane region that functions as a “signal anchor sequence” (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar, 13Nguyen M. Millar D.G. Yong V.W. Korsmeyer S.J. Shore G.C. J. Biol. Chem. 1993; 268: 25265-25268Abstract Full Text PDF PubMed Google Scholar). Integral polytopic (multispanning) OMM proteins fall into two classes, namely those that contain transmembrane β-sheets (porin) and those with α-helical hydrophobic transmembrane segments. The targeting signals for polytopic OMM proteins still have not been defined, and although limited information regarding structural determinants of the β-barrel porin is available (14Hamajima S. Sakaguchi M. Mihara K. Ono S. Sato R. J. Biochem. (Tokyo). 1988; 104: 362-367Crossref PubMed Scopus (24) Google Scholar, 15Smith M.D. Petrak M. Boucher P.D. Barton K.N. Carter L. Reddy G. Blachly-Dyson E. Forte M. Price J. Verner K. McCauley R.B. J. Biol. Chem. 1995; 270: 28331-28336Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16Court D.A. Kleene R. Neupert W. Lill R. FEBS Lett. 1996; 390: 73-77Crossref PubMed Scopus (40) Google Scholar), the precise nature of its targeting components remains unclear, as does the issue of whether porin is inserted loop by loop or in a single concerted step. Rat mitochondrial L-CPT1 and CPT2 share 50% of homology in the major part of their sequences with the exception of their N termini (17Esser 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 amino acids) ofL-CPT1 bears no significant similarity to CPT2 and contains two hydrophobic transmembrane segments (H1, residues 48–75; H2, residues 103–122) (17Esser 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, 18Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (124) Google Scholar). Both the N and C termini ofL-CPT1 are exposed to the cytosol, while the short loop connecting H1 and H2 is exposed to the intermembrane space (18Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (124) Google Scholar). Thus,L-CPT1 could be a useful model to study the mechanisms involved in targeting and membrane insertion of OMM proteins containing more than one α-helical transmembrane segment. In the present study, we have characterized the requirements forin vitro import of the rat L-CPT1 into isolated rat liver mitochondria, and by using deletion and fusion protein approaches, we have investigated the role of the N-terminal domain (residues 1–150) of L-CPT1 in mitochondrial targeting and anchoring at the OMM. In addition, through heterologous expression of several chimeric CPTs in Saccharomyces cerevisiae, we have confirmed the validity of the in vitro import results and explored the functional importance of the N-terminal domain of the ratL-CPT1. Escherichia coli DH5α strain was used to propagate various plasmids and their derivatives. The transcription plasmid pGEM4 (Promega) was used for cloning DNA fragments, for making constructs, and for in vitro transcription/translation. All of the pGEM4 constructs were under the control of the SP6 promoter. All DNA manipulations (restriction, ligation) were performed according to the instructions provided by the manufacturers' protocols of the respective enzymes. The full-length rat L-CPT1 and CPT2 cDNA inserts were retrieved from pYes2.0-CPT1 (19Brown N.F. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26438-26442Abstract Full Text PDF PubMed Google Scholar) and pBKS-CPT2 (20Woeltje K.F. Esser V. Weis B.C. Sen A. Cox W.F. McPhaul M.J. Slaughter C.A. Foster D.W. McGarry J.D. J. Biol. Chem. 1990; 265: 10720-10725Abstract Full Text PDF PubMed Google Scholar) as EcoRI and SmaI fragments, respectively, and subcloned into pGEM4. These plasmids were designated as pL-CPT1 and pCPT2. The proteins corresponding to the various constructs used in this study were created as follows. pCPT1Δ150 was generated by excising the largeBglII–EcoRI fragment of pL-CPT1 and inserted as a blunt fragment into pGEM4 cut by SmaI. This deletion construct results in loss of the first 148 N-terminal amino acids ofL-CPT1 and translation starts at the codon encoding Met151. The polymerase chain reaction (PCR) was used to copy a 377-nucleotide stretch of the mature coding sequence of CPT2 beginning at amino acid 26. The PCR was performed by using the 5′-primer (5′-GG GGT ACC AGT GCT GTC TCG GGG-3′) introducing aKpnI site and the 3′ primer (5′-C CGC TCG AGC AGT TAA ATA CAT-3′) using the unique XhoI restriction site of CPT2 cDNA. The amplified product cut by KpnI and XhoI was then inserted into pCPT2 previously digested also with KpnI and XhoI. This intermediate construct, pCPT2mat, contains the cDNA encoding the mature form of CPT2. The DNA fragment encoding the first 147 N-terminal amino acids ofL-CPT1 was generated by PCR using the sense primer (5′-CGG GTA TCA TGG CAG AGG CT-3′) containing an EcoRI site and the endogenous start ATG and the antisense primer (5′-GGG GTA CCG GTG CTG CGG CT-3′) introducing a KpnI site. The amplified DNA fragment cut by EcoRI and KpnI was ligated into pCPT2mat digested by EcoRI and KpnI. This construct encodes a chimeric protein in which the first 147 N-terminal amino acids of L-CPT1 were fused to the mature form of CPT2. Two extra amino acids (Gly-Thr) were introduced into the joining region. The cDNA encoding the first 29 amino acids of S. cerevisiae Tom70p was amplified by PCR using pOMD29 plasmid (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar) as DNA template and the following primers: the 5′-primer (5′-CGG AAT TCA TGA AGA GCT TCA TT-3′) containing an EcoRI site and the 3′-primer (5′-GGG GTA CCG TAA TAA TAG TAG GC-3′) containing a KpnI site. The amplifiedEcoRI–KpnI fragment was then ligated into pCPT2mat digested by EcoRI and KpnI. This construct encodes a fusion protein between the first N-terminal 29 amino acids of Tom70p and the mature form of CPT2. Two extra amino acids (Gly-Thr) were introduced into the joining region. The amplified PCR fragment encoding the first 29 amino acids of Tom70p was obtained by using the 5′-primer (5′-CGG AAT TCA TGA AGA GCT TCA TT-3′) containing an EcoRI site and the 3′-primer (5′-C GAA GAT CTC GTA ATA ATA GTA GGC-3′) introducing a BglII site. After digestion byEcoRI and BglII, the fragment was ligated into pGEM4CPT1Δ3′ (21Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar) cut by the same restriction enzymes. This construct encodes the 29 N-terminal amino acids of Tom70p fused toL-CPT1 lacking its first 148 amino acids. One extra amino acid (Glu) was introduced into the joining region. Plasmid pGEM4b2(167)-DHFR containing the cDNA encoding a fusion protein between the first 167 amino acids of yeast cytochrome b 2 and mouse dihydrofolate reductase (DHFR) was linearized by EcoRI and BamHI in order to remove the cytochromeb 2 component and generate pGEM4-DHFR. A fragment corresponding to the first 147 amino acids of L-CPT1 was amplified by PCR using the 5′-primer (5′-CGG AAT TCA TGG CAG AGG CT-3′) containing an EcoRI site and the 3′-primer (5′-CGG GAT CCG GTG CTG CGG CTC AT-3′) containing a BamHI site and ligated into pGEM4-DHFR as an EcoRI–BamHI fragment. The final construct encodes a fusion protein between the first 147 amino acids of L-CPT1 and the reporter protein DHFR. Four amino acids (Gly-Ser-Gly-Ile) were introduced into the joining region. The fidelity of all PCR reactions and the quality of DNA subcloning were confirmed by DNA sequence analysis. All constructs were transcribed and translated using the TNT® SP6 coupled reticulocyte lysate system according to the manufacturer's protocols (Promega) in the presence of [35S]methionine (Amersham Pharmacia Biotech, France). Lysates containing the labeled synthesized proteins were stored at −80 °C until used for the in vitro import assay. Male Wistar rats (200–300 g) bred in our laboratory were fedad libitum on a standard laboratory chow diet (62% carbohydrate, 12% fat, and 26% protein in terms of energy) with continuous access to water. 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 and then further purified on self-forming Percoll gradients (31% (w/v) Percoll) (22Zammit V.A. Corstorphine C.G. Kolodziej M.P. Biochem. J. 1989; 263: 89-95Crossref PubMed Scopus (33) Google Scholar). Protein concentration was determined by the method of Lowry et al. (23Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Purified mitochondria were resuspended in isolation buffer at a protein concentration of 20 mg/ml. Freshly isolated rat liver mitochondria (0.5 mg of protein/ml) were resuspended in import buffer (250 mm sucrose, 2 mg/ml bovine serum albumin, 20 mm Hepes-KOH, 100 mmpotassium acetate, 2 mm magnesium acetate, 2 mmdithiothreitol, 2 mm ATP, 5 mm phosphocreatine, 100 μg/ml phosphocreatine kinase, 0.4 mm GTP, 1 mm NADH, 0.6 mm spermidine, pH 7.6). After preincubation for 3 min, either at 4 or 30 °C, radiolabeled proteins (2.5%, v/v) were added, and import was performed either at 4 or 30 °C for the indicated time periods. The import reaction was stopped by dilution in 4 volumes of ice-cold KCl buffer (250 mm sucrose, 10 mm Hepes, 80 mm KCl, pH 7.6). Samples were split into two aliquots corresponding to 80 μg of mitochondrial protein, centrifuged at 12,000 × gfor 5 min at 4 °C, and washed in EDTA buffer (250 mmsucrose, 10 mm Hepes, 1 mm EDTA, pH 7.6). One of the aliquots (representing total protein binding to mitochondria) was directly analyzed on SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The other aliquot was subjected to protease treatment (in the case of CPT2 and Su9-DHFR) or carbonate extraction (in the case of OMM proteins). For protease treatment, mitochondria were resuspended in KCl buffer and treated for 15 min on ice with either 500 μg/ml (for CPT2) or 200 μg/ml (for Su9-DHFR) of trypsin (Sigma). Protease treatment was stopped by adding 5 mg/ml of soybean trypsin inhibitor (STI; Sigma). After a 10-min incubation on ice, mitochondria were washed in EDTA buffer containing STI (1 mg/ml). For carbonate extraction, mitochondria were resuspended in 0.1m Na2CO3 (pH 11.5) at a final protein concentration of 0.2 mg/ml and incubated on ice for 30 min. After centrifugation at 100,000 × g for 30 min at 4 °C, integral membrane proteins were recovered in the pellet, while soluble and peripheral proteins present in the supernatant were trichloroacetic acid-precipitated. Samples were then subjected to SDS-PAGE and exposed to Hyperfilms-MP (Amersham Pharmacia Biotech). Import rates were quantified by scanning the fluorographs. Mitochondria (5 mg/ml) were incubated on ice with 25 μg/ml of trypsin for 20 min. Then STI was added at a final concentration of 2.5 mg/ml, and mitochondria were further incubated on ice for 10 min. After centrifugation in a microcentrifuge, mitochondria were washed in EDTA buffer containing 1 mg/ml of STI, resuspended in isolation buffer at a protein concentration of 20 mg/ml, and finally used for import reactions. After a 3-min preincubation of mitochondria at 30 °C in import buffer without any energy-regenerating system, mitochondrial ATP levels were depleted by a further 10-min incubation at 30 °C in the presence of 20 μm oligomycin (Sigma). Then apyrase (40 units/ml; Sigma) was added, and mitochondria were incubated for 10 min at 30 °C before the addition of lysate. Prior to its addition to the import reaction, reticulocyte lysate was ATP-depleted by apyrase treatment for 10 min at 30 °C. For examination of the ΔΨ-dependence of import, mitochondria were incubated on ice for 5 min with 1 μm carbonyl cyanidem-chlorophenylhydrazone (CCCP; Sigma) before import. The cDNAs encoding the various chimeric proteins were retrieved from pGEM4 and subcloned into the yeast expression vector pYeDP1/8–10 (25Pompon D. Louerat B. Bronine A. Urban P. Methods Enzymol. 1996; 272: 51-64Crossref PubMed Google Scholar). First, the EcoRI–SmaI fragment encoding for CPT1-(1–147)-CPT2 was inserted into the pYeDP1/8–10 cut by the same enzymes in order to obtain the pYe-CPT1-(1–147)-CPT2 plasmid. The pOM29-CPT2 cDNA was also cloned into the pYeDP1/8–10 as an EcoRI-SmaI fragment to give the pYe-pOM29-CPT2 plasmid. The third construct designated as pYe-pOM29-CPT1Δ148 was obtained by excision from pGEM4 of theEcoRI-blunted-HindIII fragment encoding for pOM29-CPT1Δ148 and by insertion into pYeDP1/8–10 digested byEcoRI and SmaI. Each cDNA was placed under the control of the inducible GAL10 promoter and the different constructs were used to transformS. cerevisiae (haploid strain W303: MATa, his3, leu2, trp1, ura3, ade2–1, can1–100) according to Ref. 26Klebe R.J. Hariss J.V. Sharp Z.D. Dougals M.G. Gene (Amst.). 1983; 25: 333-341Crossref PubMed Scopus (372) Google Scholar. Methods for yeast culture, subcellular fractionation, and isolation of yeast mitochondria were as described previously (21Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Swelling of yeast mitochondria and Western blotting were performed as described (21Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Immunoblotting was performed as in Ref. 27Rowley N. Prip-Buus C. Westermann B. Brown C. Schwarz E. Barrell B. Neupert W. Cell. 1994; 77: 249-259Abstract Full Text PDF PubMed Scopus (202) Google Scholarusing the ECL detection system (Pierce) according to the supplier's instructions. The antisera used were against the yeast cytochromeb 2 (1:1000), the yeast mitochondrial HSP70 (1:5000), the yeast porin (1:1000), the rat L-CPT1 (1:3000) (21Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar), and the rat CPT2 (1:1000) (28Woeltje K.F. Esser V. Weis B.C. Cox W.F. Schroeder J.G. Liao S.T. Foster D.W. McGarry J.D. J. Biol. Chem. 1990; 265: 10714-10719Abstract Full Text PDF PubMed Google Scholar). CPT activity was assayed 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% bovine serum albumine (w/v) as described previously (29Herbin C. Pégorier J.P. Duée P.H. Kohl C. Girard J. Eur. J. Biochem. 1987; 165: 201-207Crossref PubMed Scopus (22) Google Scholar). Malonyl-CoA concentration was 150 μm. When the CPT assay was performed using detergent-solubilized mitochondria, mitochondria were solubilized by 5% Triton X-100 as described in Ref. 30de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar. The insoluble membrane residue was sedimented by centrifugation at 16,000 × gfor 30 min at 4 °C, and the supernatant was used for CPT assay. No CPT activity could be detected in the residual pellet fraction after solubilization. The PCR reagents and T4 DNA Polymerase were purchased from Biolabs (Ozyme, Saint-Quentin en Yvelines, France). Restriction enzymes and T4 DNA ligase were obtained from Life Technologies, Inc. Yeast culture media products were from Difco, and Zymolase 20T was from ICN Biomedicals France.l-[methyl-3H]Carnitine was from Amersham Pharmacia Biotech. Results are expressed as means ± S.E. Statistical analysis was performed using the Mann-Whitney U test. An initial series of experiments was designed to determine whetherL-CPT1 could be imported into rat liver mitochondria. The first criterion used was the tight membrane association of the nativeL-CPT1 (31Woeltje K.F. Kuwajima M. Foster D.W. McGarry J.D. J. Biol. Chem. 1987; 262: 9822-9827Abstract Full Text PDF PubMed Google Scholar). Membrane insertion of proteins can be assessed by measuring their levels of resistance to extraction with alkaline buffer (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar, 32Fujiki Y. Fowler S. Shio H. Hubbard A.L. Lazarow P. J. Biol. Biochem. 1982; 93: 103-110Google Scholar, 33Ono H. Tuboi S. Eur. J. Biochem. 1987; 168: 509-514Crossref PubMed Scopus (30) Google Scholar). This procedure distinguishes both soluble and peripherally membrane-bound proteins from integral proteins such as porin (33Ono H. Tuboi S. Eur. J. Biochem. 1987; 168: 509-514Crossref PubMed Scopus (30) Google Scholar) or Tom70p (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar). When purified rat liver mitochondria were submitted to a sodium carbonate extraction (pH 11.5), nativeL-CPT1 remained in the pellet fraction (Fig. 1 A), while CPT2 and the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (data not shown) were recovered in the supernatant fraction (Fig. 1 A). This clearly demonstrates that L-CPT1, in contrast to CPT2, is an integral mitochondrial protein and explains the different behavior of the two proteins toward detergents (31Woeltje K.F. Kuwajima M. Foster D.W. McGarry J.D. J. Biol. Chem. 1987; 262: 9822-9827Abstract Full Text PDF PubMed Google Scholar). We then asked whether once imported into freshly isolated rat liver mitochondria, only L-CPT1 could be inserted into mitochondrial membranes. Both L-CPT1 and CPT2 were synthesized in a TNT-coupled reticulocyte lysate system in the presence of [35S]methionine. After incubation with rat liver mitochondria in the import mixture, the full-length precursor CPT2 was processed into its mature form, since a protein of slightly smaller size appeared (Fig. 1 B). 90% of the mature CPT2 recovered in association with mitochondria was resistant to proteolysis even at high trypsin concentration (Fig. 1 B), whereas the CPT2 precursor present in the reticulocyte lysate was entirely digested by trypsin (data not shown). This shows that the mature CPT2 was efficiently imported into the mitochondrial matrix. Moreover, as for the native CPT2 (Fig. 1 A), the newly imported CPT2 was not inserted into the mitochondrial membranes following import, since it was completely extracted by alkaline treatment (Fig. 1 B). These results show that the assay conditions used were suitable for successful in vitro mitochondrial import of CPT proteins and that alkaline treatment of mitochondria is a useful criterion for characterization of the process. In vitrotranscription-translation of L-CPT1 cDNA leads to the synthesis of an 88-kDa 35S-labeled protein that comigrates on SDS-PAGE with the native rat L-CPT1 (data not shown). This radiolabeled L-CPT1 was then incubated in the import mixture with isolated rat liver mitochondria. The recovery ofL-CPT1 bound to mitochondria was 3-fold higher when import was performed at 30 °C rather than at 4 °C (Fig. 1 C, compare lanes 3 and 4). After import,L-CPT1 became resistant to alkaline extraction (Fig. 1 C, compare lanes 4 and 7), as was the native protein (Fig. 1 A). The existence of an alkaline-resistant form was strictly dependent upon the presence of mitochondria during import (Fig. 1 C, compare lanes 5 and 7). Thus, import of L-CPT1 leads to its insertion into mitochondrial membranes. We have checked that upon treatment of mitochondria with increasing concentrations of digitonin, both the native and the inserted radiolabeled L-CPT1 were removed from mitochondria at lower digitonin/protein ratios, whereas endogenous rat liver CPT2 still remained within the digitonin-treated mitochondria (data not shown). This implies that importedL-CPT1 was inserted into the OMM. It should be emphasized that no proteolytic cleavage could be observed upon membrane insertion of L-CPT1. Import kinetics of L-CPT1 show that membrane insertion of L-CPT1 is a time-dependent process (Fig. 1 D). Insertion was very efficient at 30 °C, since 75% of the total boundL-CPT1 was in an alkaline-resistant form after an import of 60 min (Fig. 1 D). At 4 °C, the efficiency of insertion was 7-fold lower (Fig. 1 D). These results show that upon import into rat liver mitochondria, L-CPT1 was inserted into the OMM in a temperature- and time-dependent manner. We next investigated whether the import of L-CPT1 needed the presence of the surface mitochondrial receptors (Tom complex). As a positive control for receptor dependence, we used the Su9-DHFR fusion protein, which consists of the presequence of Neurospora crassa F0-ATPase subunit 9 preceding the cytosolic mouse DHFR (34Pfanner N. Tropschug M. Neupert W. Cell. 1987; 49: 815-823Abstract Full Text PDF PubMed Scopus (192) Google Scholar). Su9-DHFR was imported into the mitochondrial matrix, where it became processed to its mature size form, which was inaccessible to exogenous added trypsin (Fig. 2A, lane 3). When mitochondria were pretreated with low concentration of trypsin to remove the mitochondrial surface receptors (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar, 33Ono H. Tuboi S. Eur. J. Biochem. 1987; 168: 509-514Crossref PubMed Scopus (30) Google Scholar), import of CPT2 and Su9-DHFR was decreased by 75 and 70%, respectively (Fig. 2 A, compare lanes 3 and 4). Insertion of L-CPT1 into trypsin-pretreated mitochondria was reduced by 60% (Fig. 2 B, compare lanes 3 and 4). Although slightly lower when compared with CPT2 and Su9-DHFR, the decrease in import efficiency of L-CPT1 due to the absence of mitochondrial receptors was similar to what was observed for other OMM proteins (12McBride H.M. Millar D.G. Li J.-M. Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (86) Google Scholar, 35Court D.A. Nargang F.E. Steiner H. Hodges R.S. N" @default.
• W1984418909 created "2016-06-24" @default.
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• W1984418909 title "The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Mediates Import into the Outer Mitochondrial Membrane and Is Essential for Activity and Malonyl-CoA Sensitivity" @default.
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