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Reconstitution of Recombinant Uncoupling Proteins

2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês

10.1074/jbc.m302126200

ISSN

1083-351X

Autores

Martin Jabůrek, Keith Garlid,

Tópico(s)

Biochemical effects in animals

Resumo

The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The K i values for ATP inhibition were 50 μm (UCP1), 70 μm (UCP2), and 120 μm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (K i = 65 μm at pH 7.2). The V max values for proton transport were also similar among the UCPs, ranging from 8 to 20 μmol·min–1·mg–1, depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport. The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The K i values for ATP inhibition were 50 μm (UCP1), 70 μm (UCP2), and 120 μm (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (K i = 65 μm at pH 7.2). The V max values for proton transport were also similar among the UCPs, ranging from 8 to 20 μmol·min–1·mg–1, depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport. Uncoupling protein 1 (UCP1) 1The abbreviations used are: UCP, uncoupling protein; BAT, brown adipose tissue; CoQ, coenzyme Q 10; FA, fatty acid; HTP, hydroxyapatite; MOPS, 3-(N-morpholino)propanesulfonic acid; sarcosyl, sodium lauroylsarcosinate; SPQ, 6-methoxy-N-(3-sulfopropyl) quinolinium; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid. of BAT mitochondria is known to dissipate energy and generate heat by catalyzing back-flux of protons into the mitochondrial matrix. This most likely occurs by a protonophoretic mechanism in which UCP transports the FA carboxylate by a flippase mechanism, and the protonated FA completes the cycle by spontaneous flip-flop through the bilayer (1Skulachev V.P. FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (395) Google Scholar, 2Garlid K.D. Orosz D.E. Modrianský M. Vassanelli S. Jezek P. J. Biol. 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Commun. 2001; 282: 273-277Crossref PubMed Scopus (119) Google Scholar, 18Arsenijevic D. Onuma H. Pecqueur C. Raimbault S. Manning B.S. Miroux B. Couplan E. Alves-Guerra M.C. Goubern M. Surwit R. Bouillaud F. Richard D. Collins S. Ricquier D. Nat. Genet. 2000; 26: 435-439Crossref PubMed Scopus (945) Google Scholar, 19Vidal-Puig A.J. Grujic D. Zhang C.-Y. Hagen T. Boss O. Ido Y. Szczepanik A. Wade J. Mootha V. Cortright R. Muoio D.M. Lowell B.B. J. Biol. Chem. 2000; 275: 16258-16266Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar). Direct evidence shows that skeletal muscle UCP3 uncouples in vivo (20Cline G.W. Vidal-Puig A.J. Dufour S. Cadman K.S. Lowell B.B. Shulman G.I. J. Biol. Chem. 2001; 276: 20240-20244Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar); however, biochemical studies have so far failed to reveal uncoupling by UCP2 and -3 in isolated mitochondria (21Cadenas S. Echtay K.S. Harper J.A. Jekabsons M.B. Buckingham J.A. Grau E. Abuin A. Chapman H. Clapham J.C. Brand M.D. J. Biol. Chem. 2002; 277: 2773-2778Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 22Couplan E. del Mar Gonzalez-Barroso M. Alves-Guerra M.C. Ricquier D. Goubern M. Bouillaud F. J. Biol. Chem. 2002; 277: 26268-26275Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Further advances in this field will require this conflict to be resolved. Our approach has been to express the UCPs in Escherichia coli followed by isolation, reconstitution, and assays of activity in liposomes. This has proven to be a useful approach for the study of mitochondrial transporters, including the oxoglutarate (23Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (183) Google Scholar), tricarboxylate (24Xu Y. Mayor J.A. Gremse D. Wood D.O. Kaplan R.S. Biochem. Biophys. Res. Commun. 1995; 207: 783-789Crossref PubMed Scopus (45) Google Scholar), citrate (25Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), phosphate (26Wohlrab H. Briggs C. Biochemistry. 1994; 33: 9371-9375Crossref PubMed Scopus (53) Google Scholar), and ADP/ATP carriers (23Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (183) Google Scholar, 27Heimpel S. Basset G. Odoy S. Klingenberg M. J. Biol. Chem. 2001; 276: 11499-11506Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We used this approach in the first demonstration of fatty acid-dependent uncoupling by UCP2 and -3 (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The anionic detergent sarcosyl was used to extract the proteins from inclusion bodies, and reconstitutively active UCP2 and -3 were obtained by dialysis of the extract in the presence of non-ionic detergent (octylpentaoxyethylene) and phospholipids. We showed that UCP2 and -3 were qualitatively identical in their transport and regulatory behavior to native UCP1. We noted, however, that the affinities of UCP2 and -3 for ATP were 5–10-fold lower than those for reconstituted, native UCP1 (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). This raised the question of whether our protocols were yielding UCPs with true native function. To address this question, we turned our attention to recombinant UCP1. The properties of native UCP1 are well established, and a successful reconstitution protocol for recombinant proteins should reproduce those properties. We now report conditions of isolation and refolding of bacterially expressed UCP1 that lead to reconstitutively active protein that behaves identically to native UCP1. Using these protocols, we show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. These experiments were carried out in the absence of coenzyme Q 10, which has been reported to be "obligatory" for UCP function (29Echtay K.S. Winkler E. Frischmuth K. Klingenberg M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1416-1421Crossref PubMed Scopus (310) Google Scholar, 30Echtay K.S. Winkler E. Klingenberg M. Nature. 2000; 408: 609-613Crossref PubMed Scopus (298) Google Scholar). We examined the effects of CoQ and its solvent, dichloromethane, on FA-catalyzed proton flux in liposomes containing UCP1, -2, and -3. We found that CoQ had no effect whatever on FA-dependent proton transport catalyzed by the UCPs or on ATP regulation of the UCPs. Dichloromethane, however, had profound effects on the liposomes, as is expected of such a strong solvent. 2A preliminary report of these results was presented in abstract form (41Jabůrek M. Jezek P. Garlid K.D. Biophys. J. 2002; 82 (Abstr. 109a)Google Scholar). Expression of UCP1, -2, and -3 in E. coli—Rat UCP1 cDNA open reading frames were amplified by PCR and inserted into the NdeI and XhoI sites of the pET21a vector (Novagen), and human UCP2 and UCP3 containing plasmids were prepared as described previously (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). From DNA sequencing, the constructs are predicted to encode proteins with amino acid sequence identical to the wild type UCP1 (31Ridley R.G. Patel H.V. Gerber G.E. Morton R.C. Freeman K.B. Nucleic Acids Res. 1986; 14: 4025-4035Crossref PubMed Scopus (63) Google Scholar), UCP2 (6Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar, 7Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1562) Google Scholar), and UCP3 (8Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (682) Google Scholar, 9Boss O. Samec S. Paoloni Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (998) Google Scholar). Plasmids were transformed into the bacterial strain BL21 (Novagen). Transformed cells were grown in the presence of carbenicillin (0.1 mg/ml) at 37 °C for several hours until A 600 reached 0.3–0.4. The expression of UCPs was induced with 1 mm isopropyl-β-d-thiogalactopyranoside, and incubation was continued at 30 °C for 3 h. Cells were lysed in a French press, and inclusion bodies were isolated and stored at –80 °C as previously described (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Extraction of UCP1, -2, and -3 from Inclusion Bodies—The pelleted inclusion bodies (about 2 mg of protein) were washed 2 times in sodium salts of 0.15 m phosphate, 25 mm EDTA, 10 mm dithiothreitol, 0.2% sodium lauroylsarcosinate (sarcosyl), pH 7.8, and centrifuged 14,000 × g for 10 min. The resulting pellet was resuspended in 4 ml of 50 mm CAPS, 25 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride, 10% glycerol, 2% sarcosyl, pH 10.0, adjusted by Tris. After 30 min of incubation at room temperature, the extract was centrifuged 14,000 × g for 10 min to remove unsolubilized particles. The supernatant was diluted rapidly in 6 ml of 10% glycerol, 1% Triton X-114, and 1 mm ATP. This mixture was incubated for 2 h at 4 °C with constant mixing. To remove sarcosyl, the extract was either dialyzed (see "Results") or passed through a 2.5-ml Dowex 11A8 column (flow rate 0.5 ml/min). The collected protein was supplemented with 5 mg/ml phosphatidylcholine and 1 mm ATP. After 2 h of incubation at 4 °C, the extract was concentrated 2-fold in an Ultrafree-15 centrifugal filter device (Millipore). 1-ml aliquots were stored at –20 °C. Purification of Recombinant UCP1, -2, and -3 on Hydroxyapatite (HTP) Column—The extracted protein was dialyzed twice for 3 h and once overnight at 1:100 dilution against the internal medium (potassium salts of 50 mm TES, 80 mm SO42- ,2mm EDTA, pH 7.5). The dialyzed protein extract was centrifuged at 14,000 × g for 10 min to remove any precipitate, and the supernatant was passed through 0.5 ml (0.12 g) of HTP (Bio-Gel column, Bio-Rad) that had been pre-equilibrated with internal medium. The flow-through was expected to contain properly folded uncoupling proteins. Isolation of UCP1 from Brown Adipose Tissue Mitochondria—UCP1 was purified and reconstituted into proteoliposomes using previously described procedures (4Jabůrek M. Varecha M. Jezek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 32Jezek P. Orosz D.E. Garlid K.D. J. Biol. Chem. 1990; 265: 19296-19302Abstract Full Text PDF PubMed Google Scholar, 33Garlid K.D. Sun X. Paucek P. Woldegiorgis G. Methods Enzymol. 1995; 260: 331-348Crossref PubMed Scopus (43) Google Scholar). Briefly, frozen BAT mitochondria were first washed with additional bovine serum albumin (5 mg/ml) and then extracted with octylpentaoxyethylene in the presence of phospholipids. UCP1 was purified on the HTP column in a medium composed of potassium salts of 50 mm TES, 25 mm SO42- , and 2 mm EDTA. The composition of phospholipids and internal medium were adjusted for subsequent reconstitution. Reconstitution of UCPs into Liposomes—Recombinant UCPs and native UCP1 were reconstituted in liposomes using previously described procedures (4Jabůrek M. Varecha M. Jezek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 33Garlid K.D. Sun X. Paucek P. Woldegiorgis G. Methods Enzymol. 1995; 260: 331-348Crossref PubMed Scopus (43) Google Scholar). Briefly, phospholipids (asolectin supplemented with 5% cardiolipin or 100% phosphatidylcholine) were dried under nitrogen and stored under vacuum overnight. The dried phospholipids were solubilized with detergent (C8E5), and protein and fluorescent probe were added. The protein/lipid mixture was incubated with Bio-Beads SM-2 (Bio-Rad) to remove detergent and form vesicles. The vesicles were passed through a Sephadex G-50–300 column to remove external probe. Protein-free liposomes were prepared using the same protocol. Fluorescence Measurements of Ion Fluxes—Ion fluxes in proteoliposomes were measured using an SLM Aminco 8000C spectrofluorometer. H+ ion fluxes were measured as changes in intraliposomal acid concentration, obtained from changes in SPQ fluorescence due to quenching by the anion of TES buffer (34Orosz D.E. Garlid K.D. Anal. Biochem. 1993; 210: 7-15Crossref PubMed Scopus (23) Google Scholar). Transport was driven by a K+ gradient initiated by the addition of 50 nm valinomycin. The proteoliposomes were studied at 0.5 mg/ml phospholipid in 2 ml of assay media at 25 °C. Each preparation was individually calibrated for fluorescence probe response, and the internal volume of vesicles was estimated from the volume of distribution of the fluorescent probe (33Garlid K.D. Sun X. Paucek P. Woldegiorgis G. Methods Enzymol. 1995; 260: 331-348Crossref PubMed Scopus (43) Google Scholar). The protein content in proteoliposomes was estimated by the Amido Black procedure (35Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar). Chemicals and Reagents—SPQ was purchased from Molecular Probes, Inc. (Eugene, OR). Asolectin (45% l-α-phosphatidylcholine) was purchased from Avanti Polar Lipids, Inc. Sulfuric acid was purchased from Fisher. All other chemicals were from Sigma. Optimizing Expression-Reconstitution Protocols for Recombinant UCPs—Our previous protocols enabled us to demonstrate that UCP2 and -3 catalyze FA-dependent, electrophoretic proton flux as well as electrophoretic flux of the FA analog, undecanesulfonate (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). However, ATP inhibition occurred with affinities that were10-fold lower than that for native UCP1 (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Because there are no standards of native activity for UCP2 and -3 in intact mitochondria, it was not possible to determine whether the altered affinities are due to biochemical differences in the protein or to artifacts arising from the method. We decided that the best way to address this question was to demonstrate that our protocols gave native function for recombinant UCP1, whose activities and regulation are well characterized. We expressed UCP1 in E. coli and carried out extraction-reconstitution using protocols adapted from those originally used for UCP2 and -3 (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Fig. 1 (squares) shows that this preparation also gave abnormally high K i values for ATP inhibition, about 480 μm. We therefore devoted our efforts to developing a protocol that would yield normal ATP affinity for bacterially expressed reconstituted UCP1. We found that the affinity for ATP improved with extended dialysis, the K i decreasing to 190 μm after a total of 44 h of dialysis (Fig. 1, circles). This finding suggested that sarcosyl may decrease the affinity of UCPs for nucleotides and that residual sarcosyl was being removed by prolonged dialysis, albeit with low efficiency. Therefore we introduced an additional treatment with a Dowex anion exchange column to improve sarcosyl removal. This approach was successful. The affinities for ATP of Dowex-treated, bacterially expressed UCP1 were nearly identical to those of native UCP1, with observed K i values of 50 μm (Fig. 1, triangles) and 65 μm (Fig. 1, crosses), respectively. We also added an HTP column purification step to meet recent criteria for isolation of bacterially expressed UCPs (36Jekabsons M.B. Echtay K.S. Brand M.D. Biochem. J. 2002; 366: 565-571Crossref PubMed Google Scholar). Based on the known behavior of native BAT UCP1, the properly folded UCPs were expected to pass through the HTP and collect in the flow-through. The recovery of protein after HTP treatment is summarized in Table I. The yield of the flow-through fraction after HTP treatment ranged from 40 to 60%. Our previous protocol (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) yielded about 20% recovery of UCP2 and UCP3 after the HTP treatment (not shown). The HTP treatment had no additional effect on the affinity of UCP1 for ATP, but it improved the apparent purity of all three UCPs, as judged by a 50% increase in the protein-specific transport rates (not shown).Table IProtein recovery after dialysis and HTP purificationStarting proteinDialyzed extractHTP flow-throughHTP recoveryμgμgμg%UCP1317 ± 54299 ± 55171 ± 4457UCP2318 ± 35279 ± 28114 ± 3441UCP3308 ± 34302 ± 34124 ± 1041 Open table in a new tab ATP Regulation of UCP1, -2, and -3—We next applied the new protocol to reconstitution of recombinant UCP2 and -3. We measured inhibition by ATP under the same conditions as those used for the UCP1 experiments of Fig. 1. Fig. 2 shows that the protocol applied to UCP2 (Fig. 2, circles) and UCP3 (Fig. 2, triangles) now yields proteins with similar affinities for ATP. In three independent experiments, the K i values were 70 μm ± 8 μm (mean ± S.D.) for UCP2, 114 μm ± 17 μm for UCP3, and 56 μm ± 8 μm for bacterially expressed UCP1, respectively. This new result shows that UCP1, -2, and -3 have similar affinities for ATP. Fatty Acid-dependent Proton Transport Mediated by UCP1, -2, and -3—It is important to note that the new protocols had no effect on H+ flux catalyzed by UCP2 and -3, and we obtained the same results as were reported previously (28Jabůrek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Our previous experiments were carried out at pH 7.2 in vesicles composed of soy bean phospholipids supplemented with 5% cardiolipin, which provide a complex lipid environment. Because we wished to evaluate the effects of CoQ, we also carried out experiments at pH 6.8 in phosphatidylcholine vesicles, conditions similar to those used by Echtay et al. (29Echtay K.S. Winkler E. Frischmuth K. Klingenberg M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1416-1421Crossref PubMed Scopus (310) Google Scholar, 30Echtay K.S. Winkler E. Klingenberg M. Nature. 2000; 408: 609-613Crossref PubMed Scopus (298) Google Scholar). The representative ion flux traces in Fig. 3 show that bacterially expressed UCP1 catalyzes FA-dependent, electrophoretic proton flux in the absence of CoQ when reconstituted in the phosphatidylcholine vesicles. Laurate induces a strong H+ flux (Fig. 3, trace a) that is sensitive to ATP (trace b), being maximally inhibited by ATP on both sides of the membrane (trace c). The partial inhibition (about 50%) by external ATP demonstrates the random orientation of the nucleotide binding sites of reconstituted UCP1, which we always observe. The transport rates with 100 μm laurate in the absence of ATP were about 10 μmol·min–1·mg–1 protein. This rate was about half of that observed with native UCP1 when flux was measured in the opposite direction (2Garlid K.D. Orosz D.E. Modrianský M. Vassanelli S. Jezek P. J. Biol. Chem. 1996; 271: 2615-2620Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). This effect is due to a smaller gradient for K+ efflux than for K+ influx. Entirely similar results were obtained with UCP2 and -3 (not shown). Importantly, these experiments, like all our previous experiments, were carried out without added CoQ. They show that CoQ is not obligatory for UCP function and confirm our published finding that bacterially expressed uncoupling proteins catalyze FA-dependent, ATP-sensitive H+ transport in the absence of CoQ. Our flux measurements are supported by extensive control experiments. We routinely measured fluxes in pure liposomes (Fig. 3, trace d), and we observed that these fluxes were nearly identical with fluxes observed in proteoliposomes containing UCP with ATP present on both sides of the membrane (Fig. 3, trace c). We also controlled for valinomycin concentration by titrating valinomycin at constant concentrations of fatty acid in both liposomes and in proteoliposomes containing UCP. This allows us to quantitate nonspecific proton transport and to choose the correct amount of valinomycin that supports the maximal ATP-sensitive rate. We find this value to be 40–80 nm (80–160 pmol·mg–1 lipid). This value is more than 20 times lower than the valinomycin concentrations used by Echtay et al. (29Echtay K.S. Winkler E. Frischmuth K. Klingenberg M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1416-1421Crossref PubMed Scopus (310) Google Scholar, 30Echtay K.S. Winkler E. Klingenberg M. Nature. 2000; 408: 609-613Crossref PubMed Scopus (298) Google Scholar). Under our experimental conditions, concentrations of valinomycin above 200 pmol·mg–1 lipid resulted in massive protein-independent fluxes, which may be due to ion pair transport of the FA anion-K+-valinomycin complex. The ATP-sensitive proton flux rates observed in our laboratory had a V max for UCP1 up to 20 μmol of H+ min–1·mg–1 protein (4Jabůrek M. Varecha M. Jezek P. Garlid K.D. J. Biol. Chem. 2001; 276: 31897-31905Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), a number that is in excellent agreement with observations in BAT mitochondria. Sundin et al. (37Sundin U. Moore G. Nedergaard J. Cannon B. Am. J. Physiol. 1987; 252: R822-R832PubMed Google Scholar) measured 42 μg of UCP1/mg of hamster BAT mitochondria. The UCP1-dependent respiration when oxidizing FA was 85 ng atoms of O min–1·mg–1 protein, which converts to 850 nmol of H+ min–1·mg–1 protein. Thus, hamster BAT mitochondria also exhibit a V max of about 20 μmol of H+ min–1·mg–1 protein. The effects of Coenzyme Q 10 on UCP1, -2, and -3 Transport and Regulation—The traces in Fig. 4 show the effects of adding CoQ dissolved in dichloromethane to the assay, as was done by Echtay et al. (29Echtay K.S. Winkler E. Frischmuth K. Klingenberg M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1416-1421Crossref PubMed Scopus (310) Google Scholar). Trace a is from a control in the absence of CoQ or dichloromethane. Trace b shows the effect of CoQ addition, which indeed caused a large increase of H+ flux. Trace c shows that an identical increase in proton flux was caused by adding the solvent without CoQ. We obtained identical results with reconstituted recombinant UCP2 and -3 (not shown). Dichloromethane is very hydrophobic and almost insoluble in water. Most of the added solvent will go into the lipid bilayer, and it is not surprising that it has profound effects. It is surprising that Echtay et al. (29Echtay K.S. Winkler E. Frischmuth K. Klingenberg M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1416-1421Crossref PubMed Scopus (310) Google Scholar) did not report any controls for solvent effects. To determine whether CoQ had any effects on UCP activity, we compared the ATP-sensitive FA-dependent H+ fluxes in the presence and absence of CoQ and dichloromethane. We varied the concentration of CoQ from 2.5 to 25 nmol·mg–1 lipid and the corresponding amount of solvent from 0.05 to 0.5% in the 2 ml of assay. As shown in Fig. 5, there was no difference between the presence (Fig. 5, filled symbols) and absence (Fig. 5, open symbols) of CoQ in these assays; all of the effect was caused by the solvent at all doses tested. Moreover, the results show that CoQ-dichloromethane caused a partial inhibition of ATP-sensitive proton flux at concentrations higher than 10 nmol CoQ·mg–1 lipid. These results show that the increased fluxes in the presence of CoQ or dichloromethane were not caused by an increased activity of the protein but most likely by a solvent-induced increase in the proton permeability of the vesicles. We further evaluated the effects of dichloromethane in the experiments of Fig. 6, which were carried out in protein-free liposomes. It can be seen that small amounts of the solvent caused large increases in the proton permeability of vesicles when FA and valinomycin were present. This liposome control of the effect of dichloromethane was also not reported in Echtay et al. (29Echtay K.S.

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