tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak.

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.

Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusion mathematical model.

The intracellular mechanisms of regulation of energy fluxes and respiration in contracting heart cells were studied. For this, we investigated the workload dependencies of the rate of oxygen consumption and metabolic parameters in Langendorff-perfused isolated rat hearts.(31)P NMR spectroscopy was used to study the metabolic changes during transition from perfusion with glucose to that with pyruvate with and without active creatine kinase system. The experimental results showed that transition from perfusion with glucose to that with pyruvate increased the phosphocreatine content and stability of its level at increased workloads. Inhibition of creatine kinase reaction by 15-min infusion of iodoacetamide decreased the maximal developed tension and respiration rates by a factor of two.(31)P NMR data were analyzed by a mathematical model of compartmentalized energy transfer, which is independent from the restrictions of the classical concept of creatine kinase equilibrium. The analysis of experimental data by this model shows that metabolic stability-constant levels of phosphocreatine, ATP and inorganic phosphate-at increased energy fluxes is an inherent property of the compartmentalized system. This explains the observed substrate specificity by changes in mitochondrial membrane potential. The decreased maximal respiration rate and maximal work output of the heart with inhibited creatine kinase is well explained by the rise in myoplasmic ADP concentration. This activates the adenylate kinase reaction in the myofibrillar space and in the mitochondria to fulfil the energy transfer and signal transmission functions, usually performed by creatine kinase. The activity of this system, however, is not sufficient to maintain high enough energy fluxes. Therefore, there is a kinetic explanation for the decreased maximal respiration rate of the heart with inhibited creatine kinase: i.e. a kinetically induced switch from an efficient energy transfer pathway (PCr-CK system) to a non-efficient one (myokinase pathway) within the energy transfer network of the cell under conditions of low apparent affinity of mitochondria to ADP in vivo. This may result in a significant decrease in the thermodynamic affinity of compartmentalized ATPase systems and finally in heart failure.

Liposomes as fatty acids carriers in isolated rat liver: effect on energy metabolism and on isolated mitochondria activity.

The effects of fatty acids (FA)-carrier, egg-lecithin liposomes (LIPO) as alternative to BSA, on ATP, glycogen and glucose contents in isolated perfused liver of fed rats were non-invasively studied using 31P/13C nuclear magnetic resonance (NMR). Oxidative phosphorylation was studied in isolated mitochondria from the same liver consecutively to the NMR experiments. ATP content decreased slowly and ATP turnover was similar during the perfusion with saline solution (KHB) or LIPO. However, LIPO induced an enhancement of respiratory control ratio in isolated mitochondria. Tissue glycogen and glucose content decreased when FA (linoleate or linolenate) were perfused with defatted BSA (3%) or LIPO (600 mg/l) whereas glucose excretion level was unchanged and lactate excretion tended to increase, reflecting changes in the cytosolic redox state and/or an enhancement of glycolysis. Addition of FA (0.5 or 1.5 mM) to LIPO caused a dramatic fall in liver ATP, a mitochondrial uncoupling and an impairment of the phosphorylation activity. Perfusion with FA (1.5 mM) carried by BSA significantly increased the ATP degradation without change of mitochondrial function. Owing to the higher affinity of BSA than LIPO for FA, these latter could be more easily released from complex LIPO-FA, increasing their uncoupling effect. Hence, the FA concentrations have to be largely decreased from the above currently used concentrations to avoid this effect. It will then be possible to minimize the effector action of FA and to study their more specific metabolic function as fuel. It was concluded that LIPO were appropriate carriers to study the different metabolic effects of FA.

Mitochondrial impairment and recovery after heat shock treatment in a human microglial cell line.

The application of a heat shock on the human microglial cell line (CHME 5) has been shown to cause cytoskeleton modifications and alterations in phosphorylated metabolite content (Macouillard-Poulletier de Gannes et al., 1998a Metabolic and cellular characterization of immortalized human microglial cells under heat stress. Neurochem. Int. 33, 61-73). In this study, we focused on the possible involvement of mitochondria in this heat stress response. The cell respiratory properties were followed during the recovering period and the possible relationships between mitochondria and the cytoskeleton were studied. We observed that the heat shock induced changes in mitochondrial activity due to protein denaturation, rather than mitochondrial loss. Furthermore, these alterations were correlated with cytoskeleton disorganization since vimentine, tubuline and mitochondria shift, simultaneously, to a perinuclear location. The perturbations of the mitochondrial distribution persisted until cytoskeleton networks had recovered. Nevertheless, the respiratory properties recovered rapidly suggesting a renaturation of mitochondrial proteins in connection with mitochondrial cytoplasmic redistribution.

Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immunoreactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

Flow cytometric analysis of mitochondrial activity in situ: application to acetylceramide-induced mitochondrial swelling and apoptosis.

Changes in mitochondrial matrix volume were studied both on isolated mitochondria and in situ on CHME 5 human microglia and monoblastoid U 937 cells using multiparametric flow cytometric analysis. The use of specific effectors of mitochondrial activity (oligomycin and KCN) allowed the demonstration, on whole cells, of a strict correlation between light scattering and mitochondrial volume changes: mitochondrial swelling induced a concomitant increase in forward scattering, and decrease in side scattering of the cell population. The technique was applied to the study of the early phases of acetyl-ceramide-induced apoptosis, which has been associated with mitochondrial dysfunction in several cellular systems. Acetyl-ceramide caused a marked swelling of isolated rat liver mitochondria. Scatter modifications were also observed in both cell lines during the first hour of incubation with acetylceramide and were accompanied by an increase in DiOC6 (3) fluorescence. The results imply that mitochondrial volume changes can be followed using flow cytometry and eventually used to assist in the interpretation of mitochondrial membrane potential variations obtained from fluorescence measurements. By applying this technique to 2 different cell lines, we demonstrated that mitochondrial swelling occurs during the early phases of acetyl-ceramide treatment, but that the induction of apoptosis is cell type-dependent.

Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A.

The purpose of this study was to test the hypothesis that mitochondrial permeability transition might be implicated in mitochondrial and intact organ dysfunctions associated with damage induced by reperfusion after cold ischaemia. Energetic metabolism was assessed continuously by 31P-NMR on a model system of isolated perfused rat liver; mitochondria were extracted from the livers and studied by using top-down control analysis. During the temperature transition from hypothermic to normothermic perfusion (from 4 to 37 degrees C) the ATP content of the perfused organ fell rapidly, and top-down metabolic control analysis of damaged mitochondria revealed a specific control pattern characterized by a dysfunction of the phosphorylation subsystem leading to a decreased response to cellular ATP demand. Both dysfunctions were fully prevented by cyclosporin A, a specific inhibitor of the mitochondrial transition pore (MTP). These results strongly suggest the involvement of the opening of MTP in vivo during the transition to normothermia on rat liver mitochondrial function and organ energetics.

Quantitative studies of enzyme-substrate compartmentation, functional coupling and metabolic channelling in muscle cells.

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics-the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.

Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition.

Upon induction of permeability transition with different agents (Ca2+, tert-butyl hydroperoxide, atractyloside), mouse hepatocyte mitochondria manifest a disruption of outer membrane integrity leading to the release of cytochrome c and apoptosis-inducing factor (AIF), two proteins which are involved in programmed cell death (apoptosis). Chelation of Ca2+ shortly (within 2 min) after its addition to isolated mitochondria reestablished the mitochondrial transmembrane potential (deltapsi(m)), prevented induction of large amplitude swelling and release of both cytochrome c and AIF. In contrast, late Ca2+ chelation (10 min after addition of Ca2+) failed to affect these parameters. Cytochrome c appears to be released through a mechanically damaged outer mitochondrial membrane rather than via a specific release mechanism. These findings clarify the mechanisms through which irreversible permeability transition occurs with subsequent large amplitude swelling culminating in the release of intermembrane proteins from mitochondria. Moreover, they confirm the hypothesis formulated by Skulachev [FEBS Lett. 397 (1996) 7-10 and Q. Rev. Biophys. 29 (1996) 169-2021 linking permeability transition to activation of the apoptogenic catabolic enzymes.

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants.

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using 31P nuclear magnetic resonance.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.

Top-down control analysis of temperature effect on oxidative phosphorylation.

The effects of temperature on the control of respiration rate, phosphorylation rate, proton leakage rate, the protonmotive force and the effective ATP/O ratio were determined in isolated rat liver mitochondria over a range of respiratory conditions by applying top-down elasticity and control analyses. Simultaneous measurements of membrane potential, oxidation and phosphorylation rates were performed under various ATP turnover rates, ranging from state 4 to state 3. Although the activities of the three subsystems decreased with temperature (over 30-fold between 37 and 4 degrees C), the effective ATP/O ratio exhibited a maximum at 25 degrees C, far below the physiological value. Top-down elasticity analysis revealed that maximal membrane potential was maintained over the range of temperature studied, and that the proton leakage rate was considerably reduced at 4 degrees C. These results definitely rule out a possible uncoupling of mitochondria at low temperature. At 4 degrees C, the decrease in ATP/O ratio is explained by the relative decrease in phosphorylation processes revealed by the decrease in depolarization after ADP addition [Diolez and Moreau (1985) Biochim. Biophys. Acta 806, 56-63]. The change in depolarization between 37 and 25 degrees C was too small to explain the decrease in ATP/O ratio. This result is best explained by the changes in the elasticity of proton leakage to membrane potential between 37 and 25 degrees C, leading to a higher leak rate at 37 degrees C for the same value of membrane potential. Top-down control analysis showed that despite the important changes in activities of the three subsystems between 37 and 25 degrees C, the patterns of the control distribution are very similar. However, a different pattern was obtained at 4 degrees C under all phosphorylating conditions. Surprisingly, control by the proton leakage subsystem was almost unchanged, although both control patterns by substrate oxidation and phosphorylation subsystems were affected at 4 degrees C. In comparison with results for 25 and 37 degrees C, at 4 degrees C there was evidence for increased control by the phosphorylation subsystem over both fluxes of oxidation and phosphorylation as well as on the ATP/O ratio when the system is close to state 3. However, the pattern of control coefficients as a function of mitochondrial activity also showed enhanced control exerted by the substrate oxidation subsystem under all intermediate conditions. These results suggest that passive membrane permeability to protons is not involved in the effect of temperature on the control of oxidative phosphorylation.

Deactivation of F0F1 ATPase in intact plant mitochondria. Effect of pH and inhibitors.

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (delta mu H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl tin and aurovertin), used at non-saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase deactivation.

Experimental discrimination between proton leak and redox slip during mitochondrial electron transport.

By measuring the relationship between protonmotive force and the increment in oxygen consumption by mitochondria treated with submaximal amounts of uncoupler, we have experimentally tested four different models of imperfect coupling of oxidative phosphorylation. The results show that the increased rate of oxygen consumption at high protonmotive force is explained entirely by the dependence on protonmotive force of the passive proton leak conductance of the mitochondrial inner membrane. There is no measurable contribution from redox-slip reactions in the proton pumps caused by high protonmotive force. Neither is there any contribution from increased proton conductance of the membrane or increased redox slip in the respiratory chain caused by high turnover rates of the complexes.

The electrochemical-proton-gradient-activated states of F0F1 ATPase in plant mitochondria as revealed by detergents.

ATP hydrolysis, triggered by the addition of polyoxyethylene-9-lauryl ether (Lubrol) or lauryldimethylamine oxide (LDAO) to energized plant mitochondria was studied in some details. The membrane disruption was quasi-instantaneous (2-3 s) with both detergents, as shown by the decrease of turbidity and the stopping of respiration. In pea leaf mitochondria, Lubrol triggered ATP hydrolysis in almost the same way as valinomycin plus nigericin, except that the activity was slightly stimulated and became insensitive to carboxyatractyloside. This allowed investigations of ATP hydrolysis without any interference of the ATP/ADP antiporter or the phosphate carrier. Lubrol did not prevent the ATPase from deactivating in pea leaf mitochondria, and did not trigger any ATP hydrolysis in potato tuber mitochondria. At variance with Lubrol, LDAO changed the properties of the F0F1 ATPase. It made the enzyme oligomycin insensitive and froze it in an activated state. The activity was also 5-8-times stimulated in pea leaf mitochondria. Moreover, LDAO revealed an important ATP hydrolase activity when added to energized potato tuber mitochondria. Despite the specific effect of LDAO, the activity triggered by this detergent strongly depended on the energized state of the organelles before detergent addition. From this study, it is concluded that the electrochemical proton gradient is completely necessary to activate the F0F1-ATPase in intact plant mitochondria, as known in chloroplasts and suggested by some reports in animal mitochondria. Moreover, it is suggested that the main difference between the enzymes of pea leaf and potato tuber mitochondria is their rate of deactivation after the collapse of the transmembrane electrochemical potential difference. Finally, when properly used, detergents appear to be a powerful tool to probe the state of the ATPase in intact mitochondria, and maybe in more integrated systems.

Tissue specificity of the regulation of ATP hydrolysis by isolated plant mitochondria.

Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a delta pH, in contrast to what was previously found for potato tuber mitochondria. This delta pH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue-specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.

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 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 significantly 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 resulted 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.