Modular assembly of yeast mitochondrial ATP synthase

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.     

Bioenergetics of the heart at high altitude: environmental hypoxia imposes profound transformations on the myocardial process of ATP synthesis

The low concentration of O₂ in the thin air at high altitude is undoubtedly the reason for the remarkable modifications in the structure and function of the heart, lung, and blood of humans permanently living under these conditions. The effect of natural hypoxia on the energy metabolism of the cell is however not well understood. Here we study the proces of ATP synthesis in the heart of guinea pigs native to high altitude (4500 m) as compared with those native to sea level. The following are the novel findings of this study. (1) The rates and extents of ATP synthesis in the presence of low concentrations of ADP (<30 M) are significantly higher at high altitude than at sea level. (2) The Hill coefficient, i.e. the degree of cooperativity between the three catalytic sites of the ATP synthase, is lower at high altitude (n = 1.36) than at sea level (n = 1.94). (3) Both, the affinity for ADP and the fractional occupancy of the catalytic sites by ATP, are higher at high altitude than at sea level but the P ₅₀, i.e. the concentration of ADP at which 50% of the catalytic sites are filled with ADP and/or ATP, is the same (74.7 M). (4) In the physiological range of ADP concentrations, the phosphorylation potential G _P is significantly higher at high altitude than at sea level. It is concluded that the molecular mechanism of energy transduction is profoundly modified at high altitude in order to readily and efficiently generate ATP in the presence of low concentrations of O₂ and ADP.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

Myocardial ischemia and in vitro mitochondrial metabolic efficiency

The purpose of this study was to evaluate the oxidative capacities and the rate of energy synthesis in isolated mitochondria extracted from normal and post-ischemic myocardium. Isolated rat hearts were perfused according to the working mode with a Krebs Heinseleit buffer containing glucose (11 mM), insulin (10 IU/1) and caprylic acid (25 M). After a 15 min perfusion in normoxic conditions, the hearts were subjected to a 20 min local zero-flow ischemia followed by a 20 min reperfusion. During the perfusion, the aortic and coronary flows, the aortic pressure and the electrocardiogram were monitored. At the end of the reperfusion period, the non-ischemic and ischemic zones (NIZ and IZ, respectively) were separated and the mitochondria were harvested from each zone. The oxygen uptake and the rate of energy production of the NIZ and IZ mitochondria were then assessed with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium concentration (0.041 and 0.150 M). Ischemia provoked a 50% reduction of coronary and aortic flows. The reperfusion of the IZ allowed the partial recovery of coronary flow, but the aortic flow decreased beneath its ischemic value because of the occurrence of severe arrhythmias, stunning and probably hibernation. The IZ mitochondria displayed a lower rate of oxygen consumption, whatever the buffer free calcium concentration. Conversely, their rate of energy production was increased, indicating that their metabolic efficiency was improved as compared to NIZ mitochondria. This might be due to the mitochondrial calcium overload persisting during reperfusion, to the activation of the inner membrane Na⁺/Ca²⁺ exchange and to a significant mitochondrial swelling. On the other hand, the presence of an elevated free calcium concentration in the respiration buffer provoked some energy wasting characterized by a constant AMP production. This was attributed to some accumulation of acetate and the activation of the energy-consuming acetylCoA synthetase. In conclusion, ischemia and reperfusion did not alter the membrane integrity of the mitochondria but improved their metabolic efficiency. Nevertheless, these in vitro results can not reflect the mitochondrial function in the reperfused myocardium. The mitochondrial calcium overload reported to last during reperfusion in the cardiomyocytes might mimic the free calcium-induced reduction of metabolic efficiency observed in vitro in the present study. The resulting energy wasting might be responsible for the contractile abnormalities noticed in the reperfused myocardium.     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

ATP hydrolysis-driven H+ translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.     

The rotary mechanism of ATP synthase

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.     

A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.     

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.     

Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe²⁺ transport by rat heart mitochondrial F₁F_o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg⁺², Ca⁺², Mn⁺², Zn⁺², Cu⁺², Fe⁺³, and Al⁺³ was observed. Moreover, Cu⁺², even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H⁺.     

Restoration of ATP synthesis in urea-treated membranes prepared from pea cotyledon mitochondria

Submitochondrial particles were prepared from pea cotyledon mitochondria by sonication in a medium containing 5 mM MgCl₂. The resulting particles (Mg²⁺-submitochondrial particles) catalyzed oxidative phosphorylation at the rate of 100–200 nmol ATP formed / min per mg protein. Treatment of Mg²⁺-submitochondrial particles with 3.0 M urea resulted in a preparation of highly resolved particles with low ATPase activity and no capacity for oxidative phosphorylation. However, the resulting membranes were not capable of reconstitution of oxidative posphorylation with the purified mitochondrial F₁-ATPase. Urea particles capable of reconstitution of oxidative phosphorylation could be prepared by extracting Mg²⁺-submitochondrial particles with concentrations of urea ranging from 1.7 to 2.0 M. We have used 1.9 M urea for large-scale preparation of urea particles that could be stored in liquid nitrogen without any loss of reconstitution capacity. The residual oxidative phosphorylation rate of these particles was 6–8 nmol ATP / min per mg protein and this rate could increase to 60–70 nmol ATP / min per mg protein on incubation with saturating amounts of purified mitochondrial F₁-ATPase. In contrast to the mitochondrial F₁, purified activated pea chloroplast CF₁ was unable to stimulate ATP synthesis in 1.9 M urea particles.     

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of ΔpH in mitochondrial ATPase-ATP synthase

Using fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlations have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of ΔpH. The results obtained are best explained in terms of local circuits of protons.     

mTOR coordinates protein synthesis,mitochondrial activity and proliferation

Protein synthesis is one of the most energy consuming processes in the cell. The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates a multitude of extracellular signals and intracellular cues to drive growth and proliferation. mTOR activity is altered in numerous pathological conditions, including metabolic syndrome and cancer. In addition to its well-established role in regulating mRNA translation, emerging studies indicate that mTOR modulates mitochondrial functions. In mammals, mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V. In this review, we highlight findings that link mTOR, mRNA translation and mitochondrial functions.     

Correlation between active form and dimeric structure of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart

The active form of purified mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated by crosslinking with dimethylsuberimidate and SDS-PAGE, with or without pretreatment with the inactivating detergent Triton X-100. In the absence of detergent, crosslinked isomers of the dimeric form of 208–235 kDa were obtained. Addition of detergent led to the simultaneous loss of the dimers and the bulk of the activity. Removal of the detergent led to a partial restoration of both activity and the dimeric forms. The results suggest that the active form is a dimer, and that the detergent-dependent conversion to the largely inactive monomer is reversible. It is proposed that the mechanism of inactivation of transhydrogenase by Triton X-100 involves a disruption of essential hydrophobic interactions between the membrane-spanning regions of the monomers.     

Tetraphenylphosphonium inhibits oxidation of physiological substrates in heart mitochondria

We show that tetraphenylphosphonium inhibits oxidation of palmitoylcarnitine, pyruvate, malate, 2-oxoglutarate and glutamate in heart mitochondria in the range of concentration (1–5 µM) commonly used for the determination of mitochondrial membrane potential. The inhibition of 2-oxoglutarate (but not other substrate) oxidation by tetraphenylphosphonium is dependent on the concentration of 2-oxoglutarate and on extramitochondrial free calcium, and the kinetic plots are consistent with a mixed type of inhibition. Our results indicate that tetraphenylphosphonium interacts with enzymes, specifically involved in the oxidation of 2-oxoglutarate, most possibly, 2-oxoglutarate dehydrogenase.     

mTOR coordinates protein synthesis,mitochondrial activity and proliferation

Protein synthesis is one of the most energy consuming processes in the cell. The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates a multitude of extracellular signals and intracellular cues to drive growth and proliferation. mTOR activity is altered in numerous pathological conditions, including metabolic syndrome and cancer. In addition to its well-established role in regulating mRNA translation, emerging studies indicate that mTOR modulates mitochondrial functions. In mammals, mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V. In this review, we highlight findings that link mTOR, mRNA translation and mitochondrial functions.