F1FO-ATPase Activity Research Articles

Novel Regioselective Synthesis of 1,3,4,5-Tetrasubstituted Pyrazoles and Biochemical Valuation on F1FO-ATPase and Mitochondrial Permeability Transition Pore Formation.

An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F1FO-ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a, 6h, and 6o on the F1FO-ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor (ESI). The dissociation constant of the ESI complex (Ki') in the presence of the 6a had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F1FO-ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o. Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a. On the contrary, 1 mM 6o had stronger desensitization of mPTP formation than 0.1 mM 6o. The F1FO-ATPase is a target of Pzs blocking mPTP formation.

The role of domain alterations in F1Fo-ATPase dysfunction associated to neurodegenerative diseases

Mitochondrial dysfunction can lead to degeneration in the central nervous system. F1Fo-ATPase catalyzes most of the intracellular ATP synthesis which plays an essential role in cellular energy supply. The dimerized assembly of F1Fo-ATPase underlies the rotational catalytic function and regulates the mechanisms of oxidative phosphorylation. F1Fo-ATPase dysfunction is involved in a variety of neurological diseases, including epilepsy, Alzheimer's disease, and Parkinson’s disease. Dysregulated expression, activity, and localization of F1Fo-ATPase subunits and the interactions with pathogenic proteins result in decreased F1Fo-ATPase activity and ATP production, and aggravated oxidative stress.

Abscisic Acid Improves Rice Thermo-Tolerance by Affecting Trehalose Metabolism.

Heat stress that occurs during the flowering stage severely decreases the rice (Oryza sativa L.) seed-setting rate. This damage can be reversed by abscisic acid (ABA), through effects on reactive oxygen species, carbohydrate metabolism, and heat shock proteins, but the exact role of trehalose and ATP in this process remains unclear. Two rice genotypes, namely, Zhefu802 (heat-resistant plant, a recurrent parent) and its near-isogenic line (faded green leaf, Fgl, heat-sensitive plant), were subjected to 38 °C heat stress after being sprayed with ABA or its biosynthetic inhibitor, fluridone (Flu), at the flowering stage. The results showed that exogenous ABA significantly increased the seed-setting rate of rice under heat stress, by 14.31 and 22.40% in Zhefu802 and Fgl, respectively, when compared with the H2O treatment. Similarly, exogenous ABA increased trehalose content, key enzyme activities of trehalose metabolism, ATP content, and F1Fo-ATPase activity. Importantly, the opposite results were observed in plants treated with Flu. Therefore, ABA may improve rice thermo-tolerance by affecting trehalose metabolism and ATP consumption.

MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites.

Endoplasmic reticulum (ER) functions critically depend on a suitable ATP supply to fuel ER chaperons and protein trafficking. A disruption of the ability of the ER to traffic and fold proteins leads to ER stress and the unfolded protein response (UPR). Using structured illumination super-resolution microscopy, we revealed increased stability and lifetime of mitochondrial associated ER membranes (MAM) during ER stress. The consequent increase of basal mitochondrial Ca2+ leads to increased TCA cycle activity and enhanced mitochondrial membrane potential, OXPHOS, and ATP generation during ER stress. Subsequently, OXPHOS derived ATP trafficking towards the ER was increased. We found that the increased lifetime and stability of MAMs during ER stress depended on the mitochondrial fusion protein Mitofusin2 (MFN2). Knockdown of MFN2 blunted mitochondrial Ca2+ effect during ER stress, switched mitochondrial F1FO-ATPase activity into reverse mode, and strongly reduced the ATP supply for the ER during ER stress. These findings suggest a critical role of MFN2-dependent MAM stability and lifetime during ER stress to compensate UPR by strengthening ER ATP supply by the mitochondria.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria.

The F1FO-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F1FO-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF1), and chloroplasts (ε and γ disulfide) emerged to block the F1FO-ATPase activity selectively. In this study, we analyze how these F1FO-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F1FO-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF1 likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria.

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction.

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

The mitochondrial F1FO-ATPase exploits the dithiol redox state to modulate the permeability transition pore

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 μM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE). Conversely, 300 μM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.

Sulfide affects the mitochondrial respiration, the Ca2+-activated F1FO-ATPase activity and the permeability transition pore but does not change the Mg2+-activated F1FO-ATPase activity in swine heart mitochondria

In mammalian cells enzymatic and non-enzymatic pathways produce H2S, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the H2S donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the F1FO-ATPase activated by the natural cofactor Mg2, but, when Mg2+ was replaced by Ca2+, a slight 15% enzyme inhibition at 100 µM NaHS was shown. Conversely, both the NADH-O2 and succinate-O2 oxidoreductase activities were totally inhibited by 200 μM NaHS with IC50 values of 61.6 ± 4.1 and 16.5 ± 4.6 μM NaHS, respectively. Since the mitochondrial respiration was equally inhibited by NaHS at both first or second respiratory substrates sites, the H2S generation may prevent the electron transfer from complexes I and II to downhill respiratory chain complexes, probably because H2S competes with O2 in complex IV, thus reducing membrane potential as a consequence of the cytochrome c oxidase activity inhibition. The Complex IV blockage by H2S was consistent with the linear concentration-dependent NADH-O2 oxidoreductase inhibition and exponential succinate-O2 oxidoreductase inhibition by NaHS, whereas the coupling between substrate oxidation and phosphorylation was unaffected by NaHS. Even if H2S is known to cause sulfhydration of cysteine residues, thiol oxidizing (GSSG) or reducing (DTE) agents, did not affect the F1FO-ATPase activities and mitochondrial respiration, thus ruling out any involvement of post-translational modifications of thiols. The permeability transition pore, the lethal channel which forms when the F1FO-ATPase is stimulated by Ca2+, did not open in the presence of NaHS, which showed a similar effect to ruthenium red, thus suggesting a putative Ca2+ transport cycle inhibition.

Mitochondrial F1FO-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.

Phenylglyoxal inhibition of the mitochondrial F1FO-ATPase activated by Mg2+ or by Ca2+ provides clues on the mitochondrial permeability transition pore

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.

Control of rotation of the F1FO-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF1 proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF1 subunits. These proteins regulate the CCW F1FO-ATPase activity by blocking γ subunit rotation at the αDP/βDP/γ subunit interface in the F1 domain. Recently, we discovered a unique natural F1-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF1, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the αDP/βDP/γ interface of the F1-ATPase. In order to exert inhibition, IF1 and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the αDP/βDP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F1FO-ATPase rotation, without affecting the CW-F1FO-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF1, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF1 inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.

Lipid-protein interactions in mitochondrial membranes from bivalve mollusks: molecular strategies in different species.

The mitochondrial F1FO-ATPase, the key enzyme in cell bioenergetics, apparently works in the same way in mollusks and in mammals. We previously pointed out a raft-like arrangement in mussel gill mitochondrial membranes, which apparently distinguishes bivalve mollusks from mammals. To explore the relationship between the microenvironmental features and the enzyme activity, the physico-chemical features of mitochondrial membranes and the F1FO-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum). Similarly to the mussel, clam gill mitochondrial membrane lipids exhibit a high sterol content (42 mg/g protein), mainly due to phytosterols (cholesterol only attains 42% of total sterols), and abundant polyunsaturated fatty acids (PUFA) (70% of total fatty acids), especially of the n-3 family. However, the F1FO-ATPase activation energies above and below the break in the Arrhenius plot (22.1 °C) are lower than in mussel and mammalian mitochondria. Laurdan fluorescence spectroscopy analyses carried out at 10 °C, 20 °C and 30 °C on mitochondrial membranes and on lipid vesicles obtained from total lipid extracts of mitochondria, indicate a physical state without coexisting domains. This mitochondrial membrane constitution, allowed by lipid-lipid and lipidprotein interactions and involving PUFA-rich phospholipids, phytosterols (much more diversified in clams than in mussels) and proteins, enables the maintenance of a homogeneous physical state in the range 10-30 °C. Consistently, this molecular interaction network would somehow extend the temperature range of the F1FO-ATPase activity and may contribute to clam resilience to temperature changes.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

Kinetic properties of the mitochondrial F1FO-ATPase activity elicited by Ca2+ in replacement of Mg2+

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg2+ or, with lower efficiency, by Ca2+. The latter may play a role when calcium concentration rises in mitochondria, a condition associated with cascade events leading to cell death. Common and distinctive features of these differently activated mitochondrial ATPases were pointed out in swine heart mitochondria. When Ca2+ replaces the natural cofactor Mg2+, the enzyme responsiveness to the transmembrane electrochemical gradient and to the classical F-ATPase inhibitors DCCD and oligomycin as well as the oligomycin sensitivity loss by thiol oxidation, are maintained. Consistently, the two mitochondrial ATPases apparently share the F1FO complex basic structure and mechanism. Peculiar cation-dependent properties, which may affect the F1 catalytic mechanism and/or the FO proton binding site features, may be linked to a different physiological role of the mitochondrial Ca-activated F-ATPase with respect to the Mg-activated F-ATPase.

Mitochondrial responses to prolonged anoxia in brain of red-eared slider turtles.

Mitochondria are central to aerobic energy production and play a key role in neuronal signalling. During anoxia, however, the mitochondria of most vertebrates initiate deleterious cell death cascades. Nonetheless, a handful of vertebrate species, including some freshwater turtles, are remarkably tolerant of low oxygen environments and survive months of anoxia without apparent damage to brain tissue. This tolerance suggests that mitochondria in the brains of such species are adapted to withstand prolonged anoxia, but little is known about potential neuroprotective responses. In this study, we address such mechanisms by comparing mitochondrial function between brain tissues isolated from cold-acclimated red-eared slider turtles (Trachemys scripta elegans) exposed to two weeks of either normoxia or anoxia. We found that brain mitochondria from anoxia-acclimated turtles exhibited a unique phenotype of remodelling relative to normoxic controls, including: (i) decreased citrate synthase and F1FO-ATPase activity but maintained protein content, (ii) markedly reduced aerobic capacity, and (iii) mild uncoupling of the mitochondrial proton gradient. These data suggest that turtle brain mitochondria respond to low oxygen stress with a unique suite of changes tailored towards neuroprotection.

Preferential nitrite inhibition of the mitochondrial F1FO-ATPase activities when activated by Ca2 + in replacement of the natural cofactor Mg2 +

BackgroundThe mitochondrial F1FO-ATP synthase has not only the known life function in building most cellular ATP, but also, as recently hinted, an amazing involvement in cell death. Accordingly, the two-faced enzyme complex, which catalyzes both ATP synthesis and ATP hydrolysis, has been involved in the mitochondrial permeability transition, the master player in apoptosis and necrosis. Nitrite, a cellular nitric oxide reservoir, has a recognized role in cardiovascular protection, through still unclear mechanisms. MethodsIn swine heart mitochondria the effect of nitrite on the F1FO-ATPase activity activated by Ca2+, henceforth defined as Ca-ATPase(s), or by the natural cofactor Mg2+, was investigated by evaluating ATP hydrolysis under different assay conditions. ResultsCa2+ is far less efficient than the natural cofactor Mg2+ in the ATPase activation. However, when activated by Ca2+ the ATPase activity is especially responsive to nitrite, which acts as uncompetitive inhibitor and up to 2mM inhibits the Ca2+-activated-ATPase(s), probably by promoting dytirosine formation on the enzyme proteins, leaving the Mg-ATPase(s) unaffected. Most likely these ATPases refer to the same F1FO complex, even if coexistent ATPases may overlap. ConclusionsThe preferential inhibition by nitrite of the Ca-ATPase(s), due to post-translational tyrosine modifications, may prevent the calcium-dependent functionality of the mitochondrial F1FO complex and related events. General significanceIn mitochondria the preferential inhibition of the Ca-ATPase activity/ies by nitrite concentrations which do not affect the coexistent Mg-ATPase(s) may quench the negative events linked to the calcium-dependent functioning mode of the F1FO complex under pathological conditions.

Lipid unsaturation per se does not explain the physical state of mitochondrial membranes in Mytilus galloprovincialis.

Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

The compound BTB06584 is an IF1‐dependent selective inhibitor of the mitochondrial F1Fo‐ATPase

Ischaemia compromises mitochondrial respiration. Consequently, the mitochondrial F1 Fo-ATPsynthase reverses and acts as a proton-pumping ATPase, so maintaining the mitochondrial membrane potential (ΔΨm ), while accelerating ATP depletion and cell death. Here we have looked for a molecule that can selectively inhibit this activity without affecting ATP synthesis, preserve ATP and delay ischaemic cell death. We developed a chemoinformatic screen based on the structure of BMS199264, which is reported to selectively inhibit F1 Fo-ATPase activity and which is cardioprotective. Results suggested the molecule BTB06584 (hereafter referred to as BTB). Fluorescence microscopy was used to study its effects on ΔΨm and on the rate of ATP consumption following inhibition of respiration in several cell types. The effect of BTB on oxygen (O2 ) consumption was explored and protective potential determined using ischaemia/reperfusion assays. We also investigated a potential mechanism of action through its interaction with inhibitor protein of F1 subunit (IF1 ), the endogenous inhibitor of the F1 Fo-ATPase. BTB inhibited F1 Fo-ATPase activity with no effect on ΔΨm or O2 consumption. ATP consumption was decreased following inhibition of respiration, and ischaemic cell death was reduced. BTB efficiency was increased by IF1 overexpression and reduced by silencing the protein. In addition, BTB rescued defective haemoglobin synthesis in zebrafish pinotage (pnt) mutants in which expression of the Atpif1a gene is lost. BTB may represent a valuable tool to selectively inhibit mitochondrial F1 Fo-ATPase activity without compromising ATP synthesis and to limit ischaemia-induced injury caused by reversal of the mitochondrial F1 Fo-ATPsynthase.

F<sub>1</sub>F<sub>O</sub> ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival(1), development and, calcium signaling(2). Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death(3). The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase(4-5). In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol(6). In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL(7) interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity(8) and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

Mitochondrial Cytochrome c Oxidase and F1Fo-ATPase Dysfunction in Peppers (Capsicum annuum L.) with Cytoplasmic Male Sterility and Its Association with orf507 and Ψatp6-2 Genes

Cytoplasmic male sterility (CMS) in pepper (Capsicum annuum L.) has been associated with novel genes in the mitochondria, such as orf507 and Ψatp6-2. Plant sterility has been proved to result from the rearrangement of the mitochondrial genome. Previous studies have demonstrated that orf507 is co-transcribed with the cox II gene, and Ψatp6-2 is truncated at the 3′ region of the atp6-2 that is found in the maintainer line. Until this time, little has been known about the relationship between the novel gene and the function of its corresponding enzyme in mitochondria from the CMS pepper line. Moreover, the aberrant function of the mitochondrial enzymes is seldom reported in pepper. In this study, we observed that anther abortion occurred after the tetrad stage in the CMS line (HW203A), which was accompanied by premature programmed cell death (PCD) in the tapetum. The spatiotemporal expression patterns of orf507 and Ψatp6-2 were analyzed together with the corresponding enzyme activities to investigate the interactions of the genes and mitochondrial enzymes. The two genes were both highly expressed in the anther. The orf507 was down-regulated in HW203A (CMS line), with nearly no expression in HW203B (the maintainer line). In contrast, the cytochrome c oxidase activity in HW203A showed the opposite trend, reaching its highest peak at the tetrad stage when compared with HW203B at the same stage. The Ψatp6-2 in the CMS line was also down-regulated, but it was up-regulated in the maintainer line. The corresponding F1Fo-ATPase activity in the CMS line was gradually decreased along with the development of the anther, which showed the same trend for Ψatp6-2 gene expression. On the contrary, with up-regulated gene expression of atp6-2 in the maintainer line, the F1Fo-ATPase activity sharply decreased after the initial development stage, but gradually increased following the tetrad stage, which was contrary to what happened in the CMS line. Taken together, all these results may provide evidence for the involvement of aberrant mitochondrial cytochrome c oxidase and F1Fo-ATPase in CMS pepper anther abortion. Moreover, the novel orf507 and Ψatp6-2 genes in the mitochondria may be involved in the dysfunction of the cytochrome c oxidase and F1Fo-ATPase, respectively, which are responsible for the abortion of anthers in the CMS line.