Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Mechanism of action of agents which uncouple oxidative phosphorylation: Direct correlation between protoncarrying and respiratory-releasing properties using rat liver mitochondria

The proton-carrying properties of uncoupling agents were investigated by measuring passive mitochondrial swelling under conditions where electrogenic proton transport was rate limiting. The ability of uncoupling agents to transport protons into mitochondria, measured in this way, was compared with respiratory stimulation. The results show that with the single exception of arsenate, all agents tested which uncouple oxidative phosphorylation demonstrate a very close correlation between release of respiration and proton transport. These findings are in support of Mitchell's original proposal that uncoupling agents act by promoting electrogenic hydrogen ion transport across the mitochondrial inner membrane.     

Altered relationship between protonmotive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid hormone status

We have determined the relationship between rate of respiration and protonmotive force in oligomycin-inhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory-chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid less than euthyroid less than hyperthyroid, while protonmotive force increased in the order hyperthyroid less than euthyroid less than hypothyroid. These findings are consistent with a thyroid-hormone-induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.     

Peroxynitrite and Brain Mitochondria: Evidence for Increased Proton Leak

Abstract: Peroxynitrite has been reported to inhibit irreversibly mitochondrial respiration. Here we show that three sequential additions of 200 µ M peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg of protein/ml) significantly stimulated state 4 respiration and that further additions progressively inhibited it. No stimulation of state 3 respiration or of the maximal enzymatic activities of the respiratory chain complexes was observed on identical peroxynitrite exposure. State 4 respiration is a consequence of the proton permeability of the mitochondrial inner membrane, and we demonstrate that the peroxynitrite-induced stimulation of state 4 respiration is accompanied by a decreased mitochondrial membrane potential, suggesting an increase in this proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. The stimulation was prevented by the lipid-soluble vitamin E analogue Trolox, suggesting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. Lipid peroxidation has previously been reported to increase membrane bilayer proton permeability. The high polyunsaturate content of brain mitochondrial phospholipids may predispose them to peroxidation, and thus a peroxynitrite-induced, lipid peroxidation-mediated increase in proton leak may apply particularly to brain mitochondria and to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.     

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.     

Regulation of mitochondrial resting state respiration: slip, leak, heterogeneity?

The contribution of molecular slippage of proton pumps, of proton leak and of coupling heterogeneity of mitochondrial population to the well-known non-linear interrelationship between resting state respiration and the protonmotive force is discussed in view of the following experimental findings. (1) After blocking mitochondrial respiration with cyanide, the rate of dissipation of the membrane potential is non-linearly dependent on the actual membrane potential, similarly to the resting state respiration in mitochondria titrated with small amounts of an inhibitor. In contrast, delta pH dissipates proportionally to its actual value. (2) The rate of electron flow from succinate to ferricyanide depends upon the protonmotive force, similarly to the flow from succinate to oxygen. This strongly suggests that the H+/e- stoichiometry in complexes III and IV of the respiratory chain is constant. (3) Mitochondria 'in situ', in permeabilized Ehrlich ascites cells, exhibit the same non-linear flux/force relationship as isolated mitochondria. These results strongly suggest that the non-ohmic characteristics of the inner mitochondrial membrane, with respect to protons driven by the membrane potential but not by the concentration gradient, is the main factor responsible for the nonlinear flux/force relationship in resting state mitochondria.     

Control of energy transformation of mitochondria. Analysis by a quantitative model

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.     

Phospholipase A activity determines the rate of respiration of the mitochondria in hibernating animals

The mechanisms for regulating the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels were studied. The microviscosity of the mitochondrial membrane in hibernating squirrels was found to be higher than that in active animals. Probably, a high microviscosity of the membrane causes a decreases in the rate of the transport of oxidation substrates into the mitochondrial matrix, which in turn may be one of the main reasons for the inhibition of mitochondrial respiration in hibernating squirrels. The activation of phospholipase A2 in a hypotonic medium results in the acceleration of the respiration and phosphorylation in the mitochondria from hibernating squirrels and is accompanied by the increase of the transport of substrates across the mitochondrial membrane. The inhibition of phospholipase A2 decreases Ca2+--induced acceleration of the transport of substrates and prevents the activation of the respiration and phosphorylation in a hypotonic medium.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

UCP1 muscle gene transfer and mitochondrial proton leak mediated thermogenesis

Mitochondrial uncoupling protein 1 (UCP1) mediates the thermogenic transport of protons through the inner mitochondrial membrane. This proton leak uncouples respiration from ATP synthesis. The current study assessed the possible contribution of UCP1 muscle gene transfer to impair mitochondrial respiration in a tissue lacking UCP1 gene expression. Rats received an intramuscular injection of plasmid pXC1 containing UCP1 cDNA in the right tibialis muscles, while left tibialis muscles were injected with empty plasmid as control. Ten days after DNA injection, mitochondria from tibialis anterior muscles were isolated and analyzed. UCP1 gene transfer resulted in protein expression as analyzed by inmunoblotting. Mitochondria isolated from UCP1-injected muscles showed a significant increase in state 2 and state 4 oxygen consumption rates and a decreased respiration control ratio in comparison to mitochondria from control muscles. Furthermore, UCP1-containing mitochondria had a lower membrane potential in those states (2 and 4) when compared with control mitochondria. Our results revealed that UCP1 muscle gene transfer is associated with an induced mitochondrial proton leak, which could contribute to increase energy expenditure.     

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.     

The relative proton stoichiometries of the mitochondrial proton pumps are independent of the proton motive force

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.     

Use the Protonmotive Force: Mitochondrial Uncoupling and Reactive Oxygen Species

Mitochondrial respiration results in an electrochemical proton gradient, or protonmotive force (pmf), across the mitochondrial inner membrane. The pmf is a form of potential energy consisting of charge (?ψ_m) and chemical (?pH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Although capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review, we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models—mitochondria and cells—only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo.     

Nature of endogenous proton conductance of the inner mitochondrial membrane. Role of Ca2+ transport system in proton transfer

In order to elucidate the nature of endogenous proton conductance of rat liver inner mitochondrial membrane, the dependence of the rate of Ca2+ transport on pH was studied. It was found that the inhibiting effect of H+ is independent of protonation of functional groups of hypothetical Ca2+ carrier, but results from electrogenic transfer of H+ across the membrane, which is highly permeable for the proton. The adsorption of H+ by mitochondria is inhibited by ruthenium red and other specific inhibitors of Ca2+ transport. It is concluded that endogenous proton conductance of the inner mitochondrial membrane depends on the functioning of the same transport system essential for membrane permeability for Ca2+ and other bivalent cations. The correlation observed between the rates of H+ and Ca2+ transport in mitochondria and the ratio of cation mobilities in aqueous solutions is in favour of a "porous" mechanism of cation transport across the mitochondrial membrane.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells

A reduce uptake and retention of the mitochondria-specific membrane potential probe rhodamine 123 by feline sarcoma virus (FeSV)-transformed mink fibroblasts (64F3) has been detected. The decreased accumulation of rhodamine 123 by 64F3 mitochondria is not due to abnormal plasma membrane dye permeability, since after microinjection of the dye these cells are still unable to retain the dye at levels comparable to the untransformed parental cells, CCL 64. Nigericin, an ionophore that mediates an electrically neutral exchange of protons for potassium ions resulting the elimination of the pH gradient across the mitochondrial membrane and a compensatory increase in mitochondrial membrane potential with continued respiration, increases both the dye uptake and the retention time in transformed 64F3 cells. These results suggest that mitochondria in FeSV-transformed mink cells may have an abnormally low mitochondrial membrane potential accompanied by a relatively high pH gradient. Since anioic metabolites such as pyruvate and glutamate are accumulated by mitochondria in proportion to the delta pH across the mitochondrial membrane, the abnormal mitochondria described here may contribute to the abnormal metabolic state of FeSV-transformed cells.     

S-D-Lactoylglutathione can be an alternative supply of mitochondrial glutathione

The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, in defending mitochondrial DNA integrity and protecting mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. To demonstrate SLG transport from cytosol to mitochondria we used radiolabeled compounds and the results showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. Also, the incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. As well mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II in inter-membrane space and mitochondrial matrix and we investigated the role of SLG in whole cells. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.     

Cardiolipins and biomembrane function.

Evidence is discussed for roles of cardiolipins in oxidative phosphorylation mechanisms that regulate State 4 respiration by returning ejected protons across and over bacterial and mitochondrial membrane phospholipids, and that regulate State 3 respiration through the relative contributions of proteins that transport protons, electrons and/or metabolites. The barrier properties of phospholipid bilayers support and regulate the slow proton leak that is the basis for State 4 respiration. Proton permeability is in the range 10(-3)-10(-4) cm s-1 in mitochondria and in protein-free membranes formed from extracted mitochondrial phospholipids or from stable synthetic phosphatidylcholines or phosphatidylethanolamines. The roles of cardiolipins in proton conductance in model phospholipid membrane systems need to be assessed in view of new findings by Hübner et al. [313]: saturated cardiolipins form bilayers whilst natural highly unsaturated cardiolipins form nonlamellar phases. Mitochondrial cardiolipins apparently participate in bilayers formed by phosphatidylcholines and phosphatidylethanolamines. It is not yet clear if cardiolipins themselves conduct protons back across the membrane according to their degree of fatty acyl saturation, and/or modulate proton conductance by phosphatidylcholines and phosphatidylethanolamines. Mitochondrial cardiolipins, especially those with high 18:2 acyl contents, strongly bind many carrier and enzyme proteins that are involved in oxidative phosphorylation, some of which contribute to regulation of State 3 respiration. The role of cardiolipins in biomembrane protein function has been examined by measuring retained phospholipids and phospholipid binding in purified proteins, and by reconstituting delipidated proteins. The reconstitution criterion for the significance of cardiolipin-protein interactions has been catalytical activity; proton-pumping and multiprotein interactions have yet to be correlated. Some proteins, e.g., cytochrome c oxidase are catalytically active when dimyristoylphosphatidylcholine replaces retained cardiolipins. Cardiolipin-protein interactions orient membrane proteins, matrix proteins, and on the outerface receptors, enzymes, and some leader peptides for import; activate enzymes or keep them inactive unless the inner membrane is disrupted; and modulate formation of nonbilayer HII-phases. The capacity of the proton-exchanging uncoupling protein to accelerate thermogenic respiration in brown adipose tissue mitochondria of cold-adapted animals is not apparently affected by the increased cardiolipin unsaturation; this protein seems to take over the protonophoric role of cardiolipins in other mitochondria. Many in vivo influences that affect proton leakage and carrier rates selectively alter cardiolipins in amount per mitochondrial phospholipids, in fatty acyl composition and perhaps in sidedness; other mitochondrial membrane phospholipids respond less or not at all.(ABSTRACT TRUNCATED AT 400 WORDS)