Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 5124-5128, July 1990 Biochemistry

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells (heterologous cell system/proton carrier/membranous transporters)

L. CASTEILLA*, 0. BLONDEL*, S. KLAUS*, S. RAIMBAULT*, P. DIOLEZt, F. MOREAUt, F. BOUILLAUD*, AND D. RICQUIER* *Centre de Recherche sur la Nutrition, Centre National de la Recherche Scientifique-Unitt Propre 1511, 9 rue Jules Hetzel, 92190 Meudon, France; and 1180, Universitd Pierre et Marie Curie, 12 rue Cuvier 75005 Paris, France

tLaboratoire de Biochimie et Biologie VWgetale, Centre National de la Recherche Scientifique-Unitd de Recherche Associde

Communicated by Pierre Joliot, April 26, 1990 (received for review January 26, 1990)

Studies on UCP offer the possibility of investigating several fundamental questions such as the mechanisms of proton transport by membrane carriers, the organization of a regulatory nucleotide binding site, the topology of a mitochondrial carrier folded inside the membrane, and lastly the targeting of a mitochondrial component with no N-terminal leader peptide. Answers to these questions require the expression of normal or mutated carriers in heterologous systems. In this work we report the expression of a functional mitochondrial carrier, UCP, in epithelial cells.

The mitochondrial uncoupling protein (UCP) ABSTRACT is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantiy lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resuited in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.

MATERIALS AND METHODS Construction of the Expression Vectors. The pECE expression vector, which contains the strong simian virus 40 (SV40) early promoter, has been described by Ellis et al. (11). The rat UCP cDNA (12) was inserted in a pTZ plasmid and the 1.2-kilobase Sst I-Sst I fragment corresponding to the complete coding sequence was inserted in the polylinker Sst I site of the dephosphorylated pECE vector. Expression vectors, designated pECE-UCP, were obtained and plasmids corresponding to sense or antisense mRNA were identified using BamHI or Bgl I digestion. Transfection of Mammalian Cells. For transient expression, pECE-UCP DNA containing normal or inverted UCP cDNA was introduced into CHO-K1 cells (10 j.g for 3 x 106 cells) by the calcium phosphate precipitation technique with a glycerol shock after 4 hr, as described by Ebina et al. (13). Stable cell lines containing the pECE-UCP cDNA were established by cotransfecting CHO cells with 15 gg of pECEUCP cDNA and 3 gg of pSV2neo DNA (14) (for 4 x 106 cells). After an 18-hr exposure to the DNA, the cells were treated with trypsin and replated at a 1:5 dilution in Ham's F12 medium (GIBCO/BRL) supplemented with glutamine (10 mM) and 10o (vol/vol) fetal calf serum. Within 48 hr, the antibiotic Geneticin (Sigma) was added to the medium at 600 gg/ml. The culture medium containing Geneticin was changed every 2 days. After 2 weeks, independent colonies were picked using 3MM paper discs saturated with trypsin and transferred to a 24-well plate. When confluent, cells were treated with trypsin and plated in 35-mm dishes. Confluent cells were then collected and analyzed using anti-UCP antibodies (15). Genomic Analysis of Stable Cells. Genomic DNA was prepared from nuclei isolated from CHO cells expressing or nonexpressing UCP. The DNA was digested with Sst I and probed with rat UCP cDNA. Immunodetection of UCP in Transfected Cells. Total protein (20-100 ,ug) or 10-50 jig of mitochondrial protein was electrophoresed in a 12% polyacrylamide gel containing NaDodSO4 and transferred to nitrocellulose by using an electric

In most eukaryotic cells, the energetic equilibrium results from processes that take place in the inner mitochondrial membrane. In this cell compartment, the synthesis of ATP is coupled to the oxidation process, so that respiration ceases when no ADP and phosphate are available. This is why two mitochondrial transporters, the ADP/ATP carrier and the phosphate carrier, play a significant role in the control of oxidative phosphorylation. These two carriers are present in any cell. The uncoupling protein (UCP) is a mitochondrial transporter (1, 2) that is expressed only in brown adipocytes of mammals, where it induces energy dissipation as heat. UCP acts as a natural uncoupler of oxidative phosphorylation capable of collapsing the proton electrochemical gradient necessary for ADP phosphorylation (1). Its activity is inhibited by purine nucleotides (1, 3, 4) and its synthesis is essentially regulated at the level of transcription by the sympathetic nervous system acting through norepinephrine released at the surface of brown adipocytes (2, 5). Interestingly, several laboratories have reported that UCP, the ADP/ATP carrier, the mitochondrial phosphate carrier, and a recently identified component named hML-7 belong to the same protein family (6-10) and were derived from a common ancestor. Moreover UCP, the ADP/ATP carrier, and hML-7 have no N-terminal targeting sequence. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: UCP, uncoupling protein; SV40, simian virus 40.

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device. Antibodies raised against rat UCP (15) were added at a 1:100 dilution and anti-sheep immunoglobulins linked to peroxidase were used as second antibody (Sigma). Isolation of CHO Cell Mitochondria. After a wash with isotonic phosphate-buffered saline, the cells were scraped, sedimented, and homogenized in 0.25 M sucrose/2 mM EDTA/10 mM potassium phosphate, pH 7.2/1% bovine serum albumin by using a motor-driven Teflon PotterElvehjem homogenizer. Usually, 4 x 108 cells were homogenized in 10 ml of medium. Nuclei and undisrupted cells were centrifuged at 800 x g for 6 min. Mitochondria were separated from the nuclear supernatant by centrifugation at 12,000 x g for 15 min and washed twice in the same medium. The final mitochondrial pellet was resuspended in the sucrose medium containing no albumin. The purity of the mitochondrial fraction and the yield of mitochondria were assessed by measuring cytochrome c oxidase activity (16). Measurement of Respiration. Mitochondrial respiration was measured with a Clark-type oxygen electrode (Hansatech system) at 250C. Mitochondrial protein (500,ug) was suspended in 1 ml of isolation medium without albumin and EDTA but with 1 mM MgCl2. Oxygen consumption was recorded continuously on a chart recorder. Measurement of Membrane Potential of CHO Cell Mitochondria. Continuous monitoring of membrane potential was performed at 250C in the respiration medium using a laboratory-constructed tetraphenylphosphonium-sensitive elecORI

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trode (17). The concentration of tetraphenylphosphonium in the medium was 5 ,uM.

RESULTS AND DISCUSSION Construction of Plasmids. The strategy used to construct a vector for the expression of rat UCP in mammalian cells is summarized in Fig. 1. The full-length UCP cDNA was inserted into the Sst I site of the pECE plasmid polylinker placed behind the SV40 early promoter. Two types of pECEUCP plasmids were produced that synthesized sense or antisense mRNA. These two types of plasmids were identified by restriction analysis. Transient Expression of the UCP in CHO Cells. CHO cells, which are known to contain a significant amount of mitochondria (18), were used. To analyze the expression of UCP in CHO cells and the insertion of UCP in mitochondria, we examined first the transient expression of pECE-UCP plasmids. CHO-K1 cells were transfected with plasmid pECEUCP5'-3' encoding UCP or with plasmid pECE-UCP3'-5' containing the inverted cDNA as a control. One example of the results obtained is shown in Fig. 2. A thin band corresponding to UCP was recognized by antibodies in the homogenate of cells transfected with plasmid pECE-UCP5'-3', and a strong signal appeared in the corresponding mitochondrial fraction. In these experiments the yield of mitochondria and the degree of purification, calculated from cytochrome c oxidase activity, were between 20 and 40% and between 6-

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FIG. 1. Construction of the rat UCP expression plasmid pECE-UCP. pECE-UCP, containing the full coding region of rat UCP was constructed with the pECE expression vector (11) and the rat UCP cDNA (12) contained in a pTZ plasmid. The Sst I-Sst I UCP cDNA contained the full coding region (solid region) and 5' and 3' untranslated domains (open regions). The large arrow indicates the 3' end of the cDNA associated with the pTZ polylinker (checked box). Two types of pECE-UCP plasmid corresponding to inverted cDNAs were obtained and identified by restriction analysis. The plasmid expressing UCP is designated pECEUCP5'-3'. Position and orientation are shown for the T7 RNA polymerase promoter, the ,B-lactamase gene conveying resistance to ampicillin (AmpR), the replication origin (ori) of the pECE plasmid, the SV40 early promoter (hatched box, PE SV40), and the poly(A) addition signal and poly(A) tract provided by the SV40 sequence (stippled box).

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FIG. 2. Transient expression of the UCP in CHO cells. Immunodetection in a mitochondrial fraction (50 gg, lanes 1 and 2) and a whole-cell homogenate (50 Lg, lanes 3 and 4). Lanes: 1 and 3, cells transfected with the pECE vector expressing antisense mRNA; 2 and 4, cells transfected with the pECE vector expressing sense mRNA; 5, 60 ,ug of mitochondrial protein of cells expressing UCP and treated with 10 ,ug of trypsin for 30 min at 370C; 6, same as lane 5 but without the addition of trypsin; 7, mitochondria (4 Ag) isolated from brown adipose tissue of cold-exposed rat; 8, molecular mass markers (Rainbow markers, Amersham). Molecular masses in kDa are shown.

mass band corresponding to the endogenous gene was also detected in wild-type and recombinant cells (lanes 3 and 4). Fig. 3 Right illustrates the immunodetection of UCP in mitochondria (10 ,ug of protein) extracted from successive plating of CHO-B5 cells. To further characterize the expression of UCP in CHO cells, UCP was purified from mitochondrial membranes of CHO-B5 cells. After their preparation with Lubrol, mitochondrial membranes were solubilized with Triton X-100 and filtered through hydroxyapatite columns (19). Solubilized membrane proteins from CHO-B5 cells contained UCP (Fig. 4, lane 3). UCP was then purified through hydroxyapatite, electrophoresed, and identified by Coomassie staining and immunodetection (Fig. 4, lanes 1 and 5). This experiment showed that UCP was really expressed in CHO cells and inserted in the mitochondrial membranes. It also indicated that its biochemical behavior was highly similar to that of brown adipocyte UCP. Subsequent experiments assessed the activity of the expressed protein. Characterization of the Functional UCP Expressed in CHO Cells. Since the action of UCP in activated brown adipocytes is to uncouple respiration and to limit ATP synthesis linked to substrate oxidation, one might expect that UCP expression could result in large phenotypic alteration and growth arrest of CHO cells. No such changes were observed in CHO-B5

and 13-fold, respectively. Thus the constructed pECE-UCP vector was functional and UCP was associated with mitochondria. Proper assembly of UCP in the mitochondria was checked using trypsin treatment of mitochondria containing UCP (Fig. 2, lane 5). Protein (4 gg) from brown fat mitochondria contains as much or more UCP than 60 ,g of mitochondrial protein extracted from transfected CHO cells, indicating that the yield of transfection of the cells was low. These results obtained with the pECE-UCP plasmid prompted us to construct stable cells expressing UCP to examine the activity of the mitochondrial carrier. Stable Expression of the UCP in CHO Cells. By using a protocol for cotransfection of CHO cells with plasmids pECE-UCP and pSVneo, we obtained several clonal cell lines that permanently expressed UCP; 85% of the colonies that survived in the selective medium containing Geneticin expressed UCP. Among these cell lines, clone B5 was selected and used in the following experiments. Nuclei were isolated from CHO-B5 cells and wild-type CHO cells, and genomic DNA was extracted. After digestion with Sst I, the DNA was electrophoresed, transferred to a nylon membrane, and probed with 32P-labeled rat UCP cDNA. The probe hybridized to a 1.2-kilobase, DNA fragment corresponding to the UCP cDNA inserted into genomic DNA from the transfected plasmid pECE-UCP (Fig. 3, lane 4). A high molecular B5

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FIG. 3. Stable expression of the UCP in CHO cells. The stable cell line expressing UCP was designated B5. Lanes 1-4 show a Southern blot analysis. Lanes: 1 and 2, nondigested genomic DNA; 3 and 4, Sst I digest of genomic DNA; 1 and 3, wild-type CHO-K1 cells; 2 and 4, CHOB5 cells expressing UCP; 5, molecular mass markers. Lanes 6-11 show immunodetection of UCP in the mitochondrial fraction (10 ug). Lanes: 6, 8, and 10, CHO-B5 cells; 7, 9, and 11, CHO-K1 cells. The various lanes correspond to successive replating of cells. Molecular masses in kDa are shown.

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FIG. 4. Purification of the UCP from CHO-B5 cells. Lanes: 1 and 2, Coomassie staining of the hydroxyapatite pass-through corresponding to solubilized mitochondrial membranes of CHO-B5 cells and wild-type cells, respectively; 3 and 4, immunodetection of UCP in Triton-solubilized membranes; 5 and 6, hydroxyapatite passthrough; 3 and 5, B5 cells; 4 and 6, K1 wild-type cells. Molecular mass corresponding to 33 kDa is shown.

cells expressing UCP. Both types of cells contained the same amount of mitochondrial components (data not shown). The measurement of large, but equivalent, amounts of lactate in culture medium of control and recombinant cells (data not shown) confirmed that such cells rely mainly on glycolysis as a source of ATP (20). However, it would be interesting to force B5 cells to use oxidative phosphorylation to synthesize ATP. To characterize the activity of UCP expressed in CHO cells, the effect of integrated UCP on mitochondrial respiration and membrane potential was investigated. In the first experiments of respiration, CHO cell mitochondria were incubated in KCI medium, as recommended for brown fat mitochondria (1). In these conditions, a low respiration rate was recorded. However, high rates of respiration were recorded when mitochondria were incubated in sucrose medium, as described for Chinese hamster fibroblasts (21). Mitochondria isolated from CHO-K1 wild-type cells exhibited a classical coupled respiration and the addition of ADP induced significant respiratory control (Fig. 5). As reported by Masini et al. (22), we observed that oligomycin strongly inhibited state-4 mitochondrial respiration. In contrast, in several experiments, we observed that mitochondria containing UCP (B5 cells, Fig. 5) exhibited loosely coupled respiration and low respiratory control (approximately half the value calculated with control mitochondria). Thus we concluded that the respiration of B5 mitochondria was partially uncoupled. One could argue that such an uncoupling was not due to expressed UCP itself but to a particular physiological state of CHO-B5 cells. To rule out this possibility, the effect of GDP addition on mitochondria was analyzed. In isolated brown fat mitochondria, GDP binds to UCP and inhibits the high proton conductance of the inner membrane leading to the restoration of coupled respiration (1). This effect of GDP is unique to cells containing UCP (1). Fig. 5 shows that, in presence of GDP, B5 mitochondria became strongly sensitive to ADP, oligomycin, or an uncoupling agent. In presence of GDP, the respiratory control observed upon addition of ADP reached the value measured with K1 control mitochondria. These experiments demonstrated that active UCP in B5 cells accounted for uncoupled respiration. We observed that 1 mM, or at least 0.5 mM, nucleotide was necessary to recouple the respiration. This is in agreement with what is known for rat and rabbit brown fat mitochondria (23). Moreover, recoupling could also be obtained with 1 mM ADP, 1 mM ATP, or 1 mM GTP whereas 1 mM AMP or 1 mM CDP was uneffective (data not shown).

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FIG. 5. Respiration of wild-type and recombinant mitochondria. One typical respiration exhibited by wild-type cell mitochondria (CHO-Ki) and two typical respirations of mitochondria containing UCP (CHO-B5) are given. Rotenone (0.5 /AM) was added to the respiration medium before addition of substrate. At the times indicated by arrows, succinate (SUC, 5 mM), ADP (0.1 mM), oligomycin (OLI, 1 Ag/IA), and 2,4-dinitrophenol (DNP, 80 ,uM) were added to the mitochondrial suspension in the oxygen electrode chamber. Respiration of mitochondria was recorded alone (upper traces) or with (lower traces) prior addition of GDP (1 mM).

The data agree with what is known on the nucleotide inhibition of UCP activity (1). In brown fat mitochondria, UCP increases the effective proton conductance of the mitochondrial inner membrane and lowers the membrane potential (1). Thus, we decided to measure the membrane potential of wild-type and recombinant mitochondria with a tetraphenylphosphonium-sensitive electrode. Results of one typical experiment are given in Fig. 6. Upon addition of succinate, a high membrane potential (193 mV) rapidly built up in control K1 mitochondria, whereas a lower membrane potential (166 mV) was slowly established in UCP-containing B5 mitochondria. This lower membrane potential can be interpreted as an increased effective proton conductance imposed by active UCP. Interestingly, addition of GDP to B5 mitochondria resulted in a rapid setting up of an improved membrane potential (180 mV) probably caused by the inhibition of the active proton translocating UCP (Fig. 6). Moreover, the values of membrane potential measured in B5 mitochondria alone or with GDP were close to values reported for brown adipose tissue mitochondria incubated in the presence or absence of GDP (24). The present work demonstrates that transfected mammalian cells expressed a functional mitochondrial carrier, UCP, and proves that uncoupling of respiration in brown adipocytes is due to UCP alone. We were also able to obtain the expression of UCP in mitochondria of Xenopus oocytes (25). However, in this system the activity of UCP could not be assessed. In CHO cells the active transporter was characterized by immunodetection, the uncoupling effect on respiration, the inhibitory effect on mitochondrial membrane potential, and the characteristic sensitivity to regulatory nucleotides. The expression of this nucleotide-sensitive proton translocator in a heterologous cell system should permit further analysis of the structure-function relationship of this membranous carrier. In particular, a strategy focused on the identification of residues involved in nucleotide binding, proton translocation, and mitochondrial targeting could be

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developed. Moreover, since the putative direct activators of UCP in brown adipocytes are free fatty acids (3, 26), the expression system of UCP in CHO cells could also be used to determine the nature of the fatty acid binding site. In other respects, expression of UCP in cells dependent on ATP produced by respiration could also be used to select cell lines expressing particular phenotypes. Finally, since UCP, the mitochondrial ADP/ATP, phosphate, and a-ketoglurate (27) carriers and hML-7 (10) share a common origin, and maybe similar membranous organization as well as particular functions, any study on mutated UCP could be informative for numerous mitochondrial transporters, for which more structural data are needed (28). We thank Dr. M. Edery for the gift of pECE expression vector, and L. Lesueur and J. Paluy for help in cell culture and cell transfection experiments. We also thank Dr. Champigny and Dr. B. Holloway for careful reading of the manuscript and P. Mdralli for expert secretarial assistance. This research was financially supported by Centre National de la Recherche Scientifique, Ministere de la Recherche et des Enseignements Supdrieurs, Institut National de la Sante et de la Recherche Mddicale, and Direction des Recherches Etudes et Technique. S.K. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.

1. Nicholls, D. G. & Locke, R. M. (1984) Physiol. Rev. 64, 1-64. 2. Ricquier, D. & Bouillaud, F. (1986) in Brown Adipose Tissue, eds. Trayhurn, P. & Nicholls, D. G. (Arnold, London), pp. 86-104. 3. Strieleman, P. J., Schalinske, K. L. & Shrago, E. (1985) J. Biol. Chem. 260, 13402-13405. 4. Klingenberg, M. & Winkler, E. (1985) EMBO J. 4, 3087-3092. 5. Ricquier, D., Bouillaud, F., Toumelin, P., Mory, G., Bazin, R., Arch, J. & Pdnicaud, L. (1986) J. Biol. Chem. 261, 1390513910. 6. Aquila, H., Link, T. M. & Klingenberg, M. (1985) EMBO J. 4, 2369-2376. 7. Bouillaud, F., Weissenbach, J. & Ricquier, D. (1986) J. Biol. Chem. 261, 1487-1490. 8. Runswick, R. J., Powell, S. J., Nyren, P. & Walker, J. E. (1987) EMBO J. 6, 1367-1373.

FIG. 6. Membrane potential of wildtype CHO-K1 mitochondria and recombinant CHO-B5 mitochondria. B5 mitochrondria contain UCP. Mitochondria (0.33 mg of protein per ml) were incubated in respiration medium containing rotenone (0.5 ,uM) at 250C. Succinate (5 mM; SUC) and 80 jM 2,4-dinitrophenol (DNP) were added where indicated. The experiments were performed in absence or presence of GDP (1 mM), which was added before the substrate. At the right, the four values correspond to exact membrane potential measured in the four recordings shown in this figure.

9. Ridley, R. G., Patel, H. V., Gerber, G. E., Morton, C. R. & Freeman, K. B. (1986) Nucleic Acids Res. 14, 4025-4035. 10. Zarilli, R., Oates, E. L., McBride, 0. W., Lerman, M. I., Chan, J. Y., Santisteban, P., Ursini, M. V., Notkins, A. L. & Kohn, L. D. (1989) Mol. Endocrinol. 3, 1498-1508. 11. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A. & Rutter, W. J. (1986) Cell 45, 721-732. 12. Bouillaud, F., Ricquier, D., Thibault, J. & Weissenbach, J. (1985) Proc. Nall. Acad. Sci. USA 82, 445-448. 13. Ebina, Y., Edery, M., Ellis, L., Standring, D., Beaudoin, J., Roth, R. A. & Rutter, W. J. (1985) Proc. Natl. Acad. Sci. USA 82, 8014-8018. 14. Southern, P. J. & Berg, P. J. (1982) Mol. Appl. Genet. 1, 327-341. 15. Ricquier, D., Barlet, J. P., Garel, J. M., Combes-George, M. & Dubois, M. P. (1983) Biochem. J. 210, 859-866. 16. Wharton, D. C. & Tzagaloff, A. (1967) Methods Enzymol. 10, 245-250. 17. Kamo, N., Muratsugu, M., Hongoh, R. & Kobatake, Y. (1979) J. Membr. Biol. 81, 127-138. 18. Yatscoff, R. W. & Freeman, K. B. (1977) Can. J. Biochem. 55, 1064-1074. 19. Ricquier, D., Lin, C. S. & Klingenberg, M. (1982) Biochem. Biophys. Res. Commun. 106, 582-589. 20. Donelly, M. & Scheffler, I. E. (1976) J. Cell. Physiol. 89, 39-52. 21. Defrancesco, L., Scheffler, I. M. & Bisell, M. J. (1976) J. Biol. Chem. 251, 4588-4595. 22. Masini, A., Cecarelli-Stanzani, D. & Muscatello, U. (1983) FEBS Lett. 160, 137-140. 23. Hohorst, H. J. & Rafael, J. (1968) Hoppe-Seyler's Z. Physiol. Chem. 349, 268-270. 24. Locke, R. M., Rial, E. & Nicholls, D. G. (1982) Eur. J. Biochem. 129, 381-387. 25. Klaus, S., Casteilla, L., Bouillaud, F., Raimbault, S. & Ricquier, D. (1990) Biochem. Biophys. Res. Commun. 167, 784789. 26. Locke, R. M., Rial, E., Scott, I. D. & Nicholls, D. G. (1982) Eur. J. Biochem. 129, 373-380. 27. Palmieri, F., Genchi, G., Zara, V., Indiveri, C. & Bisaccia, F. (1989) in Anion Carriers of Mitochondrial Membranes, eds. Azzi, A., Nalecz, K. A., Nalecz, M. J. & Wojtczak, L. (Springer, Berlin), pp. 4-15. 28. Kramer, R. & Palmieri, F. (1989) Biochem. Biophys. Acta 974, 1-23.

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was pl...
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