The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.     

Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ～ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ～ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.     

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity.     

Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F(0)F(1) ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF(1). We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F(1) sector of F(0)F(1) ATP synthase by promoting IF(1) binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF(1) binding (basal ATPase activity) or detaching bound IF(1) at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 muM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF(1) from F(0)F(1) ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF(1)-stripping conditions, indicating that diazoxide alters alkaline IF(1) release. Diazoxide inhibition of ATPase activity in IF(1)-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF(1) to F(0)F(1) ATP synthase and enhances IF(1) binding indirectly by mildly uncoupling and depolarizing mitochondria.     

In vitro and in vivo studies of F(0)F(1)ATP synthase regulation by inhibitor protein IF(1) in goat heart

A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF(1) bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF(1) content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF(1) antibodies. Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF(1) content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF(1). In addition, both in vivo and in vitro, 1.3-1.4 mol of IF(1) was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF(1) in the F(0) sector.     

Reversible assembly of the ATP-binding cassette transporter Mdl1 with the F1F0-ATP synthase in mitochondria

The half-ABC transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins. The physiological role of the peptides released from mitochondria is currently not understood. Here, we have analyzed the oligomeric state of Mdl1 in the inner membrane and demonstrate nucleotide-dependent binding to the F(1)F(0)-ATP synthase. Mdl1 forms homo-oligomeric, presumably dimeric complexes in the presence of ATP, but was found in association with the F(1)F(0)-ATP synthase at low ATP levels. Mdl1 binds membrane-embedded parts of the ATP synthase complex after the assembly of the F(1) and F(0) moieties. Although independent of Mdl1 activity, complex formation is impaired upon inhibition of the F(1)F(0)-ATP synthase with oligomycin or N,N'-dicyclohexylcarbodiimide. These results are consistent with an activation of Mdl1 upon dissociation from the ATP synthase and suggest a link of peptide export from mitochondria to the activity of the F(1)F(0)-ATP synthase and the cellular energy metabolism.     

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.     

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.     

Structural and functional characterization of F(o)F(1)-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.     

Oxidative phosphorylation in a hybrid system containing bovine heart membranes and pea mitochondrial F1-ATPase

Purified pea mitochondrial F1-ATPase reconstituted oxidative phosphorylation in both partially and completely F1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), Functional significance, and evidence for an involvement of the unique F(0) subunit F(6)

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.     

Control of mitochondrial ATP synthase in rat cardiomyocytes: effects of thyroid hormone.

Activities of the mitochondrial ATP synthase and the electron transfer chain were investigated in cultured cardiomyocytes prepared from untreated and thyroxine-treated rats. Quiescent cells from the thyroxine-treated animals showed a 33% increase in mitochondrial ATP synthase capacity, but no change in respiratory chain capacity, relative to those from control animals. This increase was attributable largely to (a) a 25% increase in F1 content in these mitochondria, and partly to (b) a 10% stimulation in ATPase activity due to raised intramitochondrial Ca2+. Both types of cell showed a normal ATP content of 38-40 nmol/mg cell protein. In control cells, the mitochondrial ATP synthase responded to increased energy demand (by electrical stimulation and/or by positive inotropic agents) with an increase in its capacity of up to 2-fold. This response was absent in cells from thyroxine-treated animals. In addition, cellular ATP levels fell significantly after 2 min electrical stimulation of cells from thyroxine-treated animals, while those of control cells were constant. It was concluded that regulation of the mitochondrial ATP synthase was defective in heart cells from thyroxine treated rats, leading to an energy deficit when energy demand on the cells was increased. Animals treated with thyroxine, but allowed to recover for 17 days after treatment, showed responses indistinguishable from the control cells. Thus, the effects of thyroxine on mitochondrial activities were reversible.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

干旱胁迫下水曲柳苗木细根线粒体的形态及活性变化

根系依赖根细胞内线粒体呼吸代谢产生的能量, 不断从土壤中获取养分。在胁迫条件下, 线粒体的结构和功能会发生一定的变化, 从而影响根系的功能。土壤干旱是最容易引起苗木细根衰老死亡的非生物胁迫因子之一。为了更好地认识干旱胁迫下细根线粒体的结构和功能变化, 对土壤干旱胁迫下水曲柳(Fraxinus mandshurica)不同颜色细根皮层薄壁细胞内线粒体的超微结构(线粒体数量、形态)、线粒体的呼吸功能、线粒体膜脂质氧化(膜透性变化、过氧化氢含量等)情况进行了研究。结果表明: (1)干旱胁迫下, 水曲柳白色及黄色根皮层薄壁细胞内线粒体形状、结构及分布数量与对照相似, 无显著差异。干旱胁迫下产生的褐色根皮层薄壁细胞线粒体数量减少, 分布密度也变小。线粒体内、外膜先后发生不同程度的解体, 最后消失。(2)干旱胁迫显著干扰了线粒体膜的正常呼吸耦联作用, 细根线粒体呼吸控制率(RCR)与磷氧比(无机磷酸/分子氧, P/O)均显著低于对照(p < 0.05)。随着细根颜色加深, 线粒体RCR和P/O值逐渐下降, 白色根﹥黄色根﹥褐色根。褐色根线粒体RCR值最低, 接近极值1。说明褐色根线粒体结构完整性最差, 能量转化效率最低。(3)干旱胁迫下, 不同颜色细根线粒体内的H₂O₂含量、线粒体膜透性、膜脂氧化产物丙二醛(MDA)含量均显著高于对照(p < 0.05)。且随着细根颜色加深, 各个值增加明显。分析可能是由于干旱胁迫导致线粒体内H₂O₂含量升高, 线粒体膜脂质过氧化(MDA含量升高), 膜结构受到破坏(膜透性增加) (电镜下可见部分线粒体内膜电子密度下降及外膜解体)。线粒体膜结构完整性的破坏, 直接影响了线粒体呼吸代谢反应, 使线粒体呼吸功能下降。     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F(0). As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N'-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F(0) channel.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.     

Modulation of nucleotide specificity of thermophilic F(o)F(1)-ATP Synthase by epsilon-subunit

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus F(o)F(1)-ATP synthase (TF(o)F(1)) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TF(o)F(1) expressed in Escherichia coli, we compared TF(o)F(1) with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TF(o)F(1) with the up-state ε was achieved by inclusion of hexokinase in the assay and TF(o)F(1) with the down-state ε was represented by εΔc-TF(o)F(1), in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TF(o)F(1) with the down-state ε synthesizes GTP at the same rate of ATP, whereas TF(o)F(1) with the up-state ε synthesizes GTP at a half-rate. Though rates are slow, TF(o)F(1) with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TF(o)F(1) from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TF(o)F(1) suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF(o)F(1).