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• W2034223813 abstract "We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 μg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 μg/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity. We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 μg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 μg/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity. uncoupling protein 1 uncoupling protein 2 uncoupling protein 3 carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone polymerase chain reaction N,N,N′,N′-tetramethyl-p-phenylenediamine methyltriphenylphosphonium selective lactate Uncoupling protein 1 (UCP1)1 uncouples brown adipose tissue mitochondria, causing physiologically important, hormonally regulated, thermogenic proton cycling across the inner membrane. The functions of the UCP1 homologues, UCP2 and UCP3 (1Fleury 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, 2Gimeno 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, 3Boss 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, 4Vidal-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), are currently uncertain (5Klingenberg M. Echtay K.S. Biochim. Biophys. Acta. 2001; 1504: 128-143Crossref PubMed Scopus (166) Google Scholar, 6Kozak L.P. Harper M.-E. Annu. Rev. Nutr. 2000; 20: 339-363Crossref PubMed Scopus (94) Google Scholar, 7Jezek P. Garlid K.D. Int. J. Biochem. Cell Biol. 1998; 30: 1163-1168Crossref PubMed Scopus (85) Google Scholar, 8Lowell B.B. Spiegelman B.M. Nature. 2000; 404: 652-660Crossref PubMed Scopus (1322) Google Scholar, 9Nedergaard J. Matthias A. Golozoubova V. Jacobsson A. Cannon B. J. Bioenerg. Biomembr. 1999; 31: 475-491Crossref PubMed Scopus (64) Google Scholar, 10Ricquier D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (756) Google Scholar, 11Brand M.D. Brindle K.M. Buckingham J.A. Harper J.A. Rolfe D.F.S. Stuart J.A. Int. J. Obes. 1999; 23 Suppl. 6: S4-S11Crossref Scopus (130) Google Scholar, 12Stuart J.A. Cadenas S. Jekabsons M.B. Roussel D. Brand M.D. Biochim. Biophys. Acta. 2001; 1504: 144-158Crossref PubMed Scopus (154) Google Scholar). They have been demonstrated to uncouple mitochondrial oxidative phosphorylation in a number of experimental models, including proteoliposomes (13Jaburek 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), yeast heterologous expression systems (1Fleury 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, 2Gimeno 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, 14Hinz W. Gruninger S. De Pover A. Chiesi M. FEBS Lett. 1999; 462: 411-415Crossref PubMed Scopus (46) Google Scholar, 15Zhang C.Y. Hagen T. Mootha V.K. Slieker L.J. Lowell B.B. FEBS Lett. 1999; 449: 129-134Crossref PubMed Scopus (102) Google Scholar, 16Rial E. Gonzalez-Barroso M. Fleury C. Iturrizaga S. Sanchis D. Jimenez-Jimenez J. Ricquier D. Goubern M. Bouillaud F. EMBO J. 1999; 18: 5827-5833Crossref PubMed Scopus (179) Google Scholar), and transgenic mice (17Clapham J.C. Arch J.R. Chapman H. Haynes A. Lister C. Moore G.B. Piercy V. Carter S.A. Lehner I. Smith S.A. Beeley L.J. Godden R.J. Herrity N. Skehel M. Changani K.K. Hockings P.D. Reid D.G. Squires S.M. Hatcher J. Trail B. Latcham J. Rastan S. Harper A.J. Cadenas S. Buckingham J.A. Brand M.D. Abuin A. Nature. 2000; 406: 415-418Crossref PubMed Scopus (520) Google Scholar). It is clear that, under some experimental conditions, heterologous or transgenic expression of these proteins can cause an increase in the proton conductance of the inner membrane (16Rial E. Gonzalez-Barroso M. Fleury C. Iturrizaga S. Sanchis D. Jimenez-Jimenez J. Ricquier D. Goubern M. Bouillaud F. EMBO J. 1999; 18: 5827-5833Crossref PubMed Scopus (179) Google Scholar, 18Cadenas S. Buckingham J.A. Clapham J.C. Brand M.D. Int. J. Obes. 2000; 24: S187Crossref Google Scholar). However, it is less obvious whether these experimental observations of uncoupling are due to a native protein activity of the UCP1 homologues, or represent a more general disruption of mitochondrial function. None of the effects observed in genetically manipulated model systems has been repeated in natural systems where changes in the levels of UCP2 and/or UCP3 occur as a response to some environmental or physiological condition (19Cadenas S. Buckingham J.A. Samec S. Seydoux J. Din N. Dulloo A.G. Brand M.D. FEBS Lett. 1999; 462: 257-260Crossref PubMed Scopus (205) Google Scholar, 20Yu X.X. Barger J.L. Boyer B.B. Brand M.D. Pan G. Adams S.H. Am. J. Physiol. 2000; 279: E433-E446PubMed Google Scholar, 21Jekabsons M.B. Gregoire F.M. Schonfeld-Warden N.A. Warden C.H. Horwitz B.A. Am. J. Physiol. 1999; 277: E380-E389PubMed Google Scholar). We have demonstrated that expression of UCP1 in yeast mitochondria can cause a nonspecific uncoupling that is not due to protein activityper se (22Stuart J.A. Harper J.A. Brindle K.M. Brand M.D. Int. J. Obes. 2000; 24: S187Crossref Scopus (21) Google Scholar, 23Stuart, J. A., Harper, J. A., Brindle, K. M., Jekabsons, M. B., and Brand, M. D. (2001) Biochem. J., in press.Google Scholar). This uncoupling artifact is present only at higher levels of UCP1 expression. At these levels, UCP1 expression in yeast also interferes with mitochondrial substrate oxidation. Similarly, Heidkaemper et al. (24Heidkaemper D. Winkler E. Muller V. Frischmuth K. Liu Q. Caskey T. Klingenberg M. FEBS Lett. 2000; 406: 1-6Google Scholar) have concluded that both UCP1 and UCP3 can be expressed in an incompetent form that interferes with ATP production. They suggest that most of the UCP3 expressed in yeast mitochondria is nonfunctional. There is considerable evidence that, under some expression regimes, a substantial proportion of the UCP1 expressed in yeast mitochondria is in fact not functional (23Stuart, J. A., Harper, J. A., Brindle, K. M., Jekabsons, M. B., and Brand, M. D. (2001) Biochem. J., in press.Google Scholar). In experiments with mammalian models, Cadenas et al. (18Cadenas S. Buckingham J.A. Clapham J.C. Brand M.D. Int. J. Obes. 2000; 24: S187Crossref Google Scholar) showed that transgenic mice overexpressing UCP3 in skeletal muscle mitochondria (17Clapham J.C. Arch J.R. Chapman H. Haynes A. Lister C. Moore G.B. Piercy V. Carter S.A. Lehner I. Smith S.A. Beeley L.J. Godden R.J. Herrity N. Skehel M. Changani K.K. Hockings P.D. Reid D.G. Squires S.M. Hatcher J. Trail B. Latcham J. Rastan S. Harper A.J. Cadenas S. Buckingham J.A. Brand M.D. Abuin A. Nature. 2000; 406: 415-418Crossref PubMed Scopus (520) Google Scholar) have lower state 3 rates of succinate oxidation. These observations suggest that UCP expression has compromised mitochondrial function in ways not related to uncoupling. This raises the question of whether the observed uncoupling following UCP2 or UCP3 expression might also be an artifact of expression and not represent a significant native activity of the protein. This is especially of concern because the amount of UCP2/UCP3 expressed in yeast mitochondria has not been quantified. Recently, information has become available regarding the levels of UCP2 that are found in mammalian mitochondria. Here we use this information, and three different yeast heterologous expression systems that yield different amounts of UCP2, to assess the effects of physiological, and supraphysiological, levels of UCP2 expression in yeast mitochondria. We relate the different levels of UCP2 expression to measured proton conductance and attempt to distinguish between native UCP2 activity and expression artifact. Human UCP2 was expressed in E. coli, where it accumulated as inclusion bodies that were subsequently harvested and used as a semipure source of UCP2 with which to calibrate UCP2 expression levels in yeast mitochondria. A PCR product for hUCP2 was made from a human mRNA library provided by Dr. Jan Digby (Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom), and its sequence was verified. It was ligated into XbaI and EcoRI restriction sites of the pET expression vector pMW172 (25Way M. Pope B. Gooch J. Hawkins M. Weeds A.G. EMBO J. 1990; 9: 4103-4109Crossref PubMed Scopus (194) Google Scholar). Competent C41 strain E. coli were transfected with either pET-UCP2 or the empty pET vector. Cultures were incubated in TB media with 100 μg/ml ampicillin at 37 °C at 250 rpm until theA600 reached 0.5–0.6. Expression of UCP2 was induced by addition of 1 mmisopropyl-μ-d-thiogalactopyranoside. After 2 h cells were harvested by centrifugation at 3000 × g for 15 min. All centrifugation steps were carried out at 4 °C. Cell pellets were stored at −85 °C. Cells were lysed in B-PER reagent (Pierce) for 10–15 min at room temperature, centrifuged at 27,200 × g for 15 min and resuspended in B-PER containing 200 μg/ml lysozyme for 5–10 min to lyse any remaining cells. Inclusion bodies were harvested by centrifugation at 27,200 × g for 15 min. The pellet was washed three times by resuspension in buffer containing 150 mm potassium phosphate, 25 mm EDTA, 1 mm dithiothreitol, 1 mm ATP, pH 7.8 (13Jaburek 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), and centrifugation at 27,200 × g. The final pellet was solubilized in 1.5% n-lauryl sarcosine for 45 min at room temperature. Insoluble material was removed by centrifugation at 27,200 × g for 15 min. The supernatant (solubilized UCP2 inclusion bodies) was stored at −85 °C. Solubilized UCP2 inclusion bodies were electrophoresed on 19-cm 12% SDS-polyacrylamide gels for 2 h at 370 V (Fig. 1 a). UCP2 content was assessed with three different stains over a range of protein loadings. For Coomassie Brilliant Blue R250 staining, protein loaded per lane was 2–20 μg (Fig. 1 a). For staining with silver (Bio-Rad) and SYPRO Orange (Bio-Rad), 0.1–1.0 μg of protein was loaded. Gels were dried overnight and then scanned using a Scanmaker 12 USL (Microtek) scanner. Band intensities were quantified using NIH Image 1.60 (available via FTP). The UCP2 content of inclusion bodies was quantified by comparing the UCP2 signal either to the signal obtained with bovine serum albumin (fraction V, assumed 90% pure) or to the total protein signal obtained within the lane. The mean estimate of the purity of the preparation used to calibrate UCP2 expression in yeast was 55 ± 7%. Mitochondrial samples and UCP2 inclusion bodies were loaded onto a 12% SDS-polyacrylamide gel and run at 160 V for 1 h in a Tris-glycine running buffer (28.8 g of glycine, 6 g of Tris in 1 liter) containing 0.1% SDS. Protein was transferred to a polyvinylidene difluoride membrane, using a Bio-Rad Trans-Blot® SD semidry electrophoretic transfer cell at 10 V for 35 min in running buffer lacking SDS and containing 20% methanol. The membrane was blocked in a phosphate-buffered saline solution containing 0.1% Tween 20 and 5% (w/v) Marvel™ nonfat dry milk powder for 1 h at room temperature. Membranes were incubated overnight at 4 °C with either of two primary antibodies. One antibody (N-19; Santa Cruz Biotechnology) was raised to a 19-amino acid epitope at the N terminus of human UCP2. It was used at a 1/1000 dilution in blocking buffer). Following several washing steps, these membranes were incubated with an alkaline phosphatase-conjugated anti-goat secondary antibody (Sigma), diluted 1/6000 in blocking buffer, for 45 min at room temperature. A second antibody (M-14; Calbiochem), raised to an epitope representing amino acids 144–157 of the mouse UCP2 sequence (which is 100% conserved between mouse and human UCP2), was also used. Membranes were incubated with a 1/2000 dilution of this primary antibody. Following several washing steps, they were exposed to an alkaline phosphatase-conjugated anti-rabbit secondary antibody (New England Biolabs) diluted 1/4000 in blocking buffer. For both antibodies, membranes were washed twice in blocking buffer and twice in a buffer containing 10 mmTris-HCl, 10 mm NaCl, 1 mm MgCl2, pH 9.5, then developed with a Phototope®-Star Western blot detection kit (New England Biolabs) and exposed for up to 1 h to Kodak X-Omat AR scientific imaging film. Films were scanned and analyzed as outlined above. UCP2 contents of yeast mitochondria were interpolated from a UCP2 inclusion body calibration series loaded on the same gel. Cells of theS. cerevisiae diploid, W303 (a/α), were transformed (26Hinnen A. Hicks J.B. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1929-1933Crossref PubMed Scopus (1375) Google Scholar) with one of three different UCP2 expression constructs. UCP2low (pBF242) was made by ligating the UCP2 PCR product intoKpnI and BamHI restriction sites of the pYES2 vector (Invitrogen), so that a 144-base untranslated region was present between the transcription start site and the initiation ATG. UCP2mid (pRUCP2) was made by ligating the UCP2 PCR product intoKpnI and SacI restriction sites of the pYES2 vector, so that a Kozak (27Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4172) Google Scholar) sequence (ACCATGG) was present at the initiation ATG. UCP2high (pBF346) was made by blunt-end ligation of the UCP2 PCR product into the pKV49 vector, so that a Kozak sequence (ATAATGG) was present at the initiation ATG. Precultures of yeast transformed with the pYES2 constructs were grown overnight in selective lactate (SL) media (28Bouillaud F. Arechaga I. Petit P.X. Raimbault S. Levi-Meyrueis C. Casteilla L. Laurent M. Rial E. Ricquier D. EMBO J. 1994; 13: 1990-1997Crossref PubMed Scopus (111) Google Scholar) (2%l-lactic acid, 0.67% yeast nitrogen base, 0.1% casamino acids, 0.12% (NH4)2SO4, 0.1% KH2PO4, 0.1% glucose, 20 mg/liter tryptophan, and 40 mg/liter adenine) to an A600 of ∼2.0, then transferred to a selective galactose medium (2%d-galactose, 0.67% yeast nitrogen base, 0.1% casamino acids, 40 mg/liter adenine, 20 mg/liter tryptophan) at 1/100 dilution for overnight (about 16 h) growth. Precultures of yeast transformed with pKV49 (pBF346 and pKV49-empty vector) were grown similarly in modified SL media, with a leucine dropout amino acid mixture (25× stock consists of, in g/liter, 0.5 adenine, 0.5 uracil, and 0.5 His, 0.75 Tyr, 0.75 Lys, 0.5 Arg, 0.5 Met, 0.75 Ile, 0.125 Phe, 0.5 Pro, 0.375 Val, 0.5 Thr, 0.875 Ser, 0.25 Glu, 0.25 Asp, 0.5 Gly, 0.5 Asn, 0.5 Ala, 0.5 Cys) in place of casamino acids. Precultures were grown to an A600 of ∼2.0 and then transferred to an identical SL medium at 1/40 dilution for overnight growth. When cultures had reached anA600 of 0.5–0.8, 1% d-galactose was added to induce expression of UCP2. Cells were harvested after 4 h (or more; see “Results”). Mitochondria were isolated following (29Guerin B. Labbe P. Somlo M. Methods Enzymol. 1979; 55: 149-159Crossref PubMed Scopus (193) Google Scholar) from yeast cultures with A600 of between 1.0 and 1.5. Yeast cells were harvested by centrifugation at 2500 × g for 5 min at room temperature, resuspended in Milli-Q grade water, recentrifuged, then resuspended in buffer containing 100 mm Tris-HCl and 20 mmdithiothreitol, pH 9.3, and incubated for 10 min at 30 °C. The cells were recentrifuged, washed twice in buffer containing 100 mm Tris-HCl and 500 mm KCl, pH 7.0, and resuspended in 5 ml of isotonic spheroplasting buffer (40 mm citric acid, 120 mm disodium hydrogen orthophosphate, 1.35 m sorbitol, 1 mm EGTA, pH 5.8). Lyticase was added at 3 mg/ml, and the cells were incubated at 30 °C for exactly 30 min. Subsequent steps were at 4 °C. Spheroplasts were pelleted, washed twice in 40 ml of buffer containing 10 mm Tris-maleate, 0.75 m sorbitol, 0.4m mannitol, 2 mm EGTA, 0.1% bovine serum albumin, pH 6.8, then resuspended in 25 ml of mitochondrial isolation buffer (30Arechaga I. Raimbault S. Prieto S. Levi-Meyrueis C. Zaragoza P. Miroux B. Ricquier D. Bouillaud F. Rial E. Biochem. J. 1993; 296: 693-700Crossref PubMed Scopus (82) Google Scholar) (0.6 m mannitol, 10 mmTris/maleate, 0.5 mm Na2HPO4, 1% bovine serum albumin, pH 6.8, with protease inhibitor tablets (Complete®; Roche Molecular Biochemicals) added at 1 tablet/40 ml immediately prior to use). The spheroplasts were homogenized by 12 passes with a Wesley Coe homogenizer. The homogenate was centrifuged at 800 × g for 10 min. The supernatants were removed by pipette, to prevent disruption of the pellet, and centrifuged at 11,000 × g for 10 min. Mitochondrial pellets were washed in buffer containing 10 mm Tris-maleate, 0.65 m mannitol, 2 mm EGTA, pH 6.8, then resuspended in a small volume of this buffer and assayed for protein content (31Gornall A.G. Bardawill C.J. David M.M. J. Biol. Chem. 1949; 177: 751-766Abstract Full Text PDF PubMed Google Scholar). Respiration was measured at 30 °C immediately following mitochondrial isolation. Mitochondria were suspended at 0.15 mg of protein/ml in 2 ml of electrode buffer (10 mm Tris/maleate, 0.6 m mannitol, 0.5 mm EGTA, 2 mm MgCl2, 10 mm K2HPO4, 0.1% bovine serum albumin, pH 6.8) containing 3 mm NADH in a Rank oxygen electrode. Respiratory control ratios (rate with FCCP/rate without) for control mitochondria were around 7 (Table II), comparable to published studies (1Fleury 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, 14Hinz W. Gruninger S. De Pover A. Chiesi M. FEBS Lett. 1999; 462: 411-415Crossref PubMed Scopus (46) Google Scholar, 16Rial E. Gonzalez-Barroso M. Fleury C. Iturrizaga S. Sanchis D. Jimenez-Jimenez J. Ricquier D. Goubern M. Bouillaud F. EMBO J. 1999; 18: 5827-5833Crossref PubMed Scopus (179) Google Scholar). Additions were made as solutions in water (NADH, GDP), or methanol (FCCP, fatty acids).Table IIRespiration with NADH as substrate in mitochondria isolated from yeast containing UCP2 expression constructsNADHNADH + FCCPRCRUCP2low165 ± 231311 ± 2457.9 ± 0.8Paired control161 ± 241256 ± 2027.8 ± 0.4UCP2mid290 ± 641849 ± 3176.9 ± 0.6Paired control284 ± 391878 ± 1516.8 ± 0.4UCP2high501 ± 542-150Significantly different from paired control (p < 0.01).1351 ± 812.8 ± 0.12-150Significantly different from paired control (p < 0.01).Paired control260 ± 341522 ± 1576.0 ± 0.4Values are in nmol of O/min/mg of mitochondrial protein, and represent means ± S.E. of five to seven separate experiments with two or three different transformants for each construct. Respiratory control ratio (RCR) = (NADH + FCCP rate)/NADH rate.2-150 Significantly different from paired control (p < 0.01). Open table in a new tab Values are in nmol of O/min/mg of mitochondrial protein, and represent means ± S.E. of five to seven separate experiments with two or three different transformants for each construct. Respiratory control ratio (RCR) = (NADH + FCCP rate)/NADH rate. Complex III was inhibited with myxothiazol, and ascorbate in the presence of the artificial electron carrier N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was used as a well defined respiratory substrate whose oxidation could be titrated conveniently. The oxygen electrode was fitted with a methyltriphenylphosphonium (TPMP)-sensitive electrode to allow simultaneous measurements of membrane potential (32Brand M.D. Brown G.C. Cooper C.E. Bioenergetics: A Practical Approach. Oxford University Press, New York1995: 39-62Google Scholar) and oxygen consumption rate (which is equal to proton leak rate divided by the H+/O ratio of 4.0). The dependence of oxygen consumption rate on membrane potential gives the kinetic response of the proton leak to its driving force. The proton conductance at each membrane potential can be read from the proton leak curves. Mitochondria were suspended in electrode buffer at 0.5 mg/ml, 30 °C. Oligomycin (1 μg/ml) was added to inhibit the ATP synthase, so that all oxygen consumption was attributable to the leak pathway and not ATP synthesis. Nigericin (100 ng/ml) was added to clamp the pH gradient across the inner membrane. Myxothiazol (3 μm) was added to inhibit electron transport at respiratory complex III because some additions were in ethanol, which can be oxidized by yeast mitochondria through an NADH-linked pathway. 2 mm ascorbate was used as respiratory substrate. Ascorbate oxidation is through cytochromec oxidase and is linearly dependent on TMPD, which catalyzes electron transport to mitochondrial cytochrome c. The TPMP electrode was then calibrated with four additions, each of 1 μm TPMP. Ascorbate oxidation was increased sequentially by adding TMPD to cumulative concentrations of 6.25, 12.25, 25, 37.5, 50, and 75 μm. At each TMPD concentration, steady-state oxygen consumption and membrane potential were measured. Membrane potentials were calculated from TPMP concentrations outside the mitochondria, as described in Ref. 32Brand M.D. Brown G.C. Cooper C.E. Bioenergetics: A Practical Approach. Oxford University Press, New York1995: 39-62Google Scholar, assuming a TPMP binding correction of 0.4 (μl/mg)−1. A different TPMP binding correction would affect all the measured values of membrane potential but would not significantly affect our conclusions. Means were compared using Student'st test. Chemicals were purchased from Sigma, unless otherwise stated. The levels of UCP2 expression in mitochondria isolated from transfected yeast were determined by Western blot using solubilized UCP2 inclusion bodies as calibration standards (Fig. 1). The two antibodies (C-14 and N-19) gave virtually identical results. UCP2 was expressed in yeast mitochondria at three levels that ranged from 33 ng/mg of mitochondrial protein in UCP2low yeast to 4 μg/mg in UCP2high yeast (Table I).Table IUCP2 expression levels in mitochondria isolated from yeast containing UCP2 expression constructsYeast expression constructInduction regimeUCP2 expression level(ng UCP2/mg mitochondrial protein)UCP2lowOvernight 2%d-galactose32.5 ± 3.3UCP2midOvernight 2% d-galactose283 ± 22UCP2high4-h 1% d-galactose4066 ± 1274UCP2 concentrations were determined using N-19 antibody calibrated with inclusion body UCP2 (see “Experimental Procedures”). Values represent means ± S.E. from three to five separate experiments with two or three transformants for each construct. Open table in a new tab UCP2 concentrations were determined using N-19 antibody calibrated with inclusion body UCP2 (see “Experimental Procedures”). Values represent means ± S.E. from three to five separate experiments with two or three transformants for each construct. Induction of UCP2 expression in UCP2low yeast had no effect on yeast growth rates. The mean doubling time of UCP2low yeast in selective galactose medium was 1.83 ± 0.07 h, compared with 1.81 ± 0.03 h (S.E., n = 6) in paired controls grown under identical conditions. Respiration rates with NADH as substrate, both coupled and uncoupled with FCCP, were not different between UCP2low mitochondria and their paired controls (Table II). Palmitate (50 μm) stimulated respiration equally in UCP2low and control mitochondria (data not shown). The proton leak kinetics were determined in mitochondria isolated from UCP2low yeast and paired controls (Fig.2 a). Over the range of membrane potentials (driving force for proton leak), no differences in proton conductance were observed. Induction of UCP2 expression in UCP2mid yeast had no effect on growth. The doubling time of UCP2mid yeast growing in exponential phase following induction was 1.75 ± 0.06 h, compared with 1.77 ± 0.06 (S.E., n = 6) for paired controls. Respiration rates with NADH as substrate, both coupled and uncoupled with FCCP, were not different in mitochondria isolated from UCP2mid yeast and paired controls (Table II). Palmitate (50 μm) and all-trans-retinoic acid (45 μm, buffer, pH 7.3) stimulated respiration equally in UCP2mid and control mitochondria (Fig. 3, a andb). The proton leak kinetics of UCP2mid mitochondria were similar to control mitochondria (Fig. 2 b). UCP2mid mitochondria had identical, or perhaps slightly lower, proton conductance than controls at all measured values of membrane potential. Induction of UCP2 expression in UCP2high yeast significantly inhibited growth rate in the exponential phase. The doubling time of UCP2high yeast in exponential phase following induction with 1% d-galactose was 4.1 ± 0.1 h, compared with 2.6 ± 0.1 h (S.E., n = 7) for paired controls. Mitochondria isolated from UCP2high yeast had significantly higher rates of respiration with NADH as substrate, slightly (not significantly) lowered FCCP uncoupled rates and significantly lowered respiratory control (Table II and Fig. 3 c). GDP did not inhibit respiration with NADH as substrate in UCP2high mitochondria or their paired controls (Fig. 3 c). 3 mm GDP slightly stimulated respiration in UCP2high mitochondria (Fig.3 c), perhaps through the nucleotide inducible proton conductance pathway (33Prieto S. Bouillaud F. Rial E. Arch. Biochem. Biophys. 1996; 334: 43-49Crossref PubMed Scopus (28) Google Scholar). Our assay conditions were designed to minimize the proton leak through this pathway (33Prieto S. Bouillaud F. Rial E. Arch. Biochem. Biophys. 1996; 334: 43-49Crossref PubMed Scopus (28) Google Scholar), but may not have abolished it entirely. UCP2high yeast mitochondria had altered proton leak kinetics (Fig.2 c). At all measured membrane potentials, proton conductance was greater in UCP2high mitochondria. Membrane potentials and oxygen consumption rates can be compared for each concentration of TMPD; UCP2high mitochondria achieved a lower membrane potential, but did not respire faster than control mitochondria. Increased proton conductance normally lowers membrane potential and stimulates respiration (34Brand M.D. Biochim. Biophys. Acta. 1990; 1018: 128-133Crossref PubMed Scopus (232) Google Scholar). Thus, substrate (ascorbate/TMPD) oxidation was impaired in UCP2high mitochondria. Indeed, fully FCCP-uncoupled rates of NADH oxidation in UCP2high mitochondria became progressively impaired as the time between UCP2 induction and mitochondrial isolation increased (Fig.4). This was also apparent when the oxidized substrate was ascorbate/TMPD (data not shown). Antibodies to UCP2 are typically able to detect the presence of the protein expressed in yeast mitochondria, but the same antibodies often fail to detect UCP2 in mitochondria from mammalian tissues. Recently, this has been shown to be due to two limitations: low levels of UCP2 in mitochondria from most mammalian tissues, and cross-reactivity of the commercially available UCP2 antibodies with other proteins with apparent molecular masses of about 32 kDa. Nonetheless, UCP2 levels in mammalian mitochondria have recently been quantified, using an antibody whose specificity has been verified using mitochondria from the tissues of wild-type and" @default.
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• W2034223813 title "Physiological Levels of Mammalian Uncoupling Protein 2 Do Not Uncouple Yeast Mitochondria" @default.
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