Localized energization of the mitochondrial inner membrane by ATP.

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane. The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F1 . F0) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211-222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F1), reflecting the localized energization of the membrane by ATP.     

Localized energization of the mitochondrial inner membrane by ATP

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane.The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F₁ · F_o) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211–222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F₁), reflecting the localized energization of the membrane by ATP.     

The insensitivity to uncouplers of testis mitochondrial ATPase

Albumin-free testis mitochondrial ATPase activity failed to be stimulated by either 2,4-dinitrophenol (DNP) or carbonyl cyanide rho-trifluoromethoxyphenylhydrazone (FCCP). DNP scarcely enhanced the state 4 respiration and mitochondria proved to be poorly coupled. When 1% bovine serum albumin was added to the isolation medium, DNP or FCCP stimulated ATPase nearly twofold and the dose-response curves for the uncouplers on the QO2 reached a plateau at five- to sixfold. The DNP coupling index (q) also showed a 30-40% improvement. A dose-response curve for oligomycin on the rate of [gamma-32P]ATP synthesis showed a stimulation of ATP synthase activity by 10-100 ng inhibitor/mg protein, suggesting a possible blockade of "open" F0 channels. In the albumin preparation oligomycin inhibited ATP synthesis in the range 10-100 ng/mg protein. Since testis ATPase is known to be loosely bound to the membrane, an effect of albumin, improving tightness in the interaction of the F1 and the F0 sectors of the ATPase, is suggested.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Mitochondrial adenosine triphosphatase of wild-type and poky Neurospora crassa.

We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Km for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 +/- 0.03 mumol/min-mg of protein compared with 1.33 +/- 0.07 mumol/min-mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 +/- 0.2 mumol/min-mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky.     

ATP synthase complex from bovine heart mitochondria: the oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase.

The requirement of bovine heart mitochondrial oligomycin sensitivity conferring protein (OSCP) in conferring dicyclohexylcarbodiimide (DCCD)-sensitivity to membrane-bound F1 was investigated by using OSCP-depleted membrane fraction (UF0) of ATP synthase. The ATPase activity of UF0-F1 was completely insensitive to DCCD while that of UF0-F1-OSCP was inhibited 95% by 16 microM DCCD. Both UF0-F1 and UF0-F1-OSCP complexes bound 5 nmol [14C]DCCD/mg UF0, and all the radioactivity was found to be associated with the DCCD-binding proteolipid. The data suggest that OSCP may be necessary for transmitting not only energy-linked signals, but also signals induced by F0 inhibitory ligands in mitochondrial energy transduction.     

Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.     

The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA

Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Influence of adenine nucleotides on oligomycin inhibition of energy-transducing reactions in intact rat-liver mitochondria.

The inhibitory effect of oligomycin was investigated in intact mitochondria through oxidative phosphorylation and uncoupler induced ATPase activity. Results show that oligomycin inhibition curves can be either sigmoidal or hyperbolic depending on experimental conditions and chiefly on the metabolic state of mitochondria with regard to the distribution of mitochondrial endogenous adenine-nucleotides. Active respiration and uncoupler-induced ATPse activity produce sigmoidal titration curves for a high initial ATP : ADP ratio and hyperbolic curves for a low ATP : ADP ratio. Time-dependent inhibitions are observed for the two reactions. The maximal inhibitory action for low concentrations of the inhibitor is delayed by the initial presence of ATP or the possibility of generating from inorganic phosphate before adding oligomycin. Results presented here show that the initial adenine-nucleotide distribution is important for oligomycin sensitivity of energy-linked reactions. Although a limited conformational change of the oligomycin-sensitivity to the inhibitor, it is more likely that a gross structural change of the inner membrane induced by adenine-nucleotides modifies membrane permeability to oligomycin.     

Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.     

31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase

The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the plasma membrane ATPase of Saccharomyces cerevisiae

1. The distribution of ATPase and several marker enzymes was examined after differential and sucrose gradient centrifugation of yeast homogenates. 2. An ATPase activity not sensitive to oligomycin is found exclusively associated with a particulate fraction equilibrating at densities of 1.23-1.25. This particulate material shows the chemical and enzymatic characteristics of the yeast plasma membrane. 3. The pH optimum of the plasma membrane ATPase is 5.6, as compared with 8.5 for the mitochondrial ATPase. In addition to oligomycin, the enzyme is not sensitive to other inhibitors of the mitochondrial ATPase as azide, dicyclohexylcarbodiimide and the mitochondrial ATPase inhibitor protein. It is inhibited by p-chloromercuryphenyl sulfonate, fluoride, quercetin and by the antibiotic Dio-9 but is not affected by ouabain. 4. The plasma membrane ATPase shows a high affinity for ATP (Km = 0.1 mM) and is very specific for this compound, hydrolyzing other nucleotide triphosphates less than 25% as rapidly. No activity was detected with ADP. 5. The enzyme requires a divalent cation for activity and Mg2+ is the most effective. It is not significantly stimulated by K+ or bicarbonate and Ca2+ is inhibitory. 6. The activity cannot be assayed in intact cells unless they are permeabilized with toluene. This suggest that the active site is on the cytoplasmic side of the plasma membrane.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Effect of mitochondrial energetic state on radiation-induced DNA internucleosomal fragmentation in irradiated thymocytes

One of the earliest features of apoptosis is a decrease of mitochondrial membrane potential. Here we show that when apoptosis is induced in thymocytes by ionizing radiation, inhibitors of mitochondrial energy production suppress DNA internucleosomal fragmentation. The suppression of fragmentation by inhibitors does not correlate with their effect on mitochondrial membrane potential, as it was observed when membrane potential was decreased (in the presence of inhibitors of respiratory chain, uncouplers of oxidative phosphorylation) or non affected (in the presence of oligomycin, inhibitor of mitochondrial ATPase).     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.