Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria

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 microg/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 microgram/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.     

An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

Membrane protein import in yeast mitochondria

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn(2+)-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous intermembrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.     

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

Background  

Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle.     

Uncoupling protein 1 affects the yeast mitoproteome and oxygen free radical production

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.     

Lactate utilization in mitochondria prevents Bax cytotoxicity in yeast Kluyveromyces lactis

In a search for the physiological conditions able to suppress the disruption of electron transport through the inner mitochondrial membrane induced by Bax, we found that respiratory substrate - lactate completely abolished Bax toxicity in yeast Kluyveromyces lactis. The effect of lactate was dependent on the presence of cytochrome c, as no effect was observed in the cytochrome c null strain. The investigation of lactate effect on markers of Bax toxicity showed that: (i) oxidation of lactate did not affect the decrease in oxygen consumption, but (ii) lactate was able to diminish the generation of reactive oxygen species and simultaneously to suppress Bax-induced cell death. We show that suppression of Bax lethality in K. lactis can be, in addition to anti-apoptotic proteins, achieved also by the utilization of lactate in the mitochondria.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

Endogenous free radical generation may influence proteolysis in mitochondria

Isolated Mitochondria were allowed to incorporate radioactive amino acids into protein and proteolysis was then measured. In State 4 free radical generation was manipulated by means of respiratory chain blockers and uncouplers. Conditions of enhanced radical flux resulted in accelerated protein breakdown. We suggest that radicals influence proteolysis in cells both directly (by fragmenting proteins) and indirectly (by rendering proteins more susceptible to proteinases).     

Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.     

Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.     

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.     

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.     

Estrogen prevents oxidative damage to the mitochondria in Friedreich's ataxia skin fibroblasts

Estrogen and estrogen-related compounds have been shown to have very potent cytoprotective properties in a wide range of disease models, including an in vitro model of Friedreich's ataxia (FRDA). This study describes a potential estrogen receptor (ER)-independent mechanism by which estrogens act to protect human FRDA skin fibroblasts from a BSO-induced oxidative insult resulting from inhibition of de novo glutathione (GSH) synthesis. We demonstrate that phenolic estrogens, independent of any known ER, are able to prevent lipid peroxidation and mitochondrial membrane potential (ΔΨm) collapse, maintain ATP at near control levels, increase oxidative phosphorylation and maintain activity of aconitase. Estrogens did not, however, prevent BSO from depleting GSH or induce an increased expression level of GSH. The cytoprotective effects of estrogen appear to be due to a direct overall reduction in oxidative damage to the mitochondria, enabling the FRDA fibroblast mitochondria to generate sufficient ATP for energy requirements and better survive oxidative stress. These data support the hypothesis that phenol ring containing estrogens are possible candidate drugs for the delay and/or prevention of FRDA symptoms.     

Energy conservation and uncoupling in mitochondria

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of M_r = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.     

Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system.

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8-fold, while uncoupling protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type uncoupling protein 1. Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity. However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.