F1 Part Research Articles

Leukemic cells resist lysosomal inhibition through the mitochondria-dependent reduction of intracellular pH and oxidants.

Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5-80μM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24h. Leukemic cells produced more acidic organelles to tolerate 5-10μM CQ. CQ (20-80μM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80μM CQ challenge, co-treatment with the inhibitor of F0 part of mitochondrial H+-ATP synthase, oligomycin (40nM), prevented the elevation of oxidants as well as pHi, and attenuated stresses on mitochondria, cell survival, and cell proliferation. Besides, oligomycin-treated cells obviously displayed the lysosomal peripheralization and plasma membrane blebbing, suggesting that these cells were in process of lysosomal exocytosis and microvesicle release. Enhanced motion of these secretory processes allowed the cells to exclude CQ and repair necrotic injury. Together, the oxidant production and the proton dynamic interconnection among lysosomes, mitochondria, and cytosol are crucial for leukemic susceptibility to lysosomotropic chemotherapeutics.

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans.

In most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), β (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of S. lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as S. coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and the previous published results of our group, we propose an entirely novel conception of the nature of this link.

Cofactor Engineering Redirects Secondary Metabolism and Enhances Erythromycin Production in Saccharopolyspora erythraea.

Saccharopolyspora erythraea is used for industrial erythromycin production. To explore the physiological role of intracellular energy state in metabolic regulation by S. erythraea, we initially overexpressed the F1 part of the endogenous F1F0-ATPase in the high yielding erythromycin producing strain E3. The F1-ATPase expression resulted in lower [ATP]/[ADP] ratios, which was accompanied by a strong increase in the production of a reddish pigment and a decreased erythromycin production. Subsequent transcriptional analysis revealed that the lower intracellular [ATP]/[ADP] ratios exerted a pleotropic regulation on the metabolism of S. erythraea. The lower [ATP]/[ADP] ratios induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or regenerate NADH. The F1-ATPase overexpression strain exhibited a state of redox stress, which was correlated to an alteration of electron transport at the branch of the terminal oxidases, and S. erythraea channeled the enhanced glycolytic flux toward a reddish pigment in order to reduce NADH formation. The production of erythromycin was decreased, which is in accordance with the net ATP requirement and the excess NADH formed through this pathway. Partial growth inhibition by apramycin increased the intracellular [ATP]/[ADP] ratios and demonstrated a positive correlation between [ATP]/[ADP] ratios and erythromycin synthesis. Finally, overexpression of the entire F1F0-ATPase complex resulted in 28% enhanced erythromycin production and markedly reduced pigment synthesis in E3. The work illustrates a feasible strategy to optimize the distribution of fluxes in secondary metabolism.

F0F1 ATP synthase regulates extracellular calcium influx in human neutrophils by interacting with Cav2.3 and modulates neutrophil accumulation in the lipopolysaccharide-challenged lung

BackgroundNeutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown.MethodsBlue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo.ResultsWe found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and β subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Cav2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Cav2.3 functional complex indicated that it can regulate extracellular Ca2+ influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage.ConclusionsIn this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target.Graphical abstract

Towards unidirectional reconstitution of membrane proteins into liposomes

Functional studies of membrane proteins (MP) in their native environment (the cellular membrane) is challenging due to the complexity of the native membranes and measurement interferences from other membrane constituents or reactions. Therefore, MPs are often purified and integrated into systems which mimic their natural environment in a membrane as e.g. liposomes or nanodiscs, a process called reconstitution, allowing to investigate its function and structural aspects without any disturbing background. The most important problem during MP-reconstitution is the often random orientation of the MP in the liposomal membrane after reconstitution. For functional studies of the MP of interest and quantitative analysis of its properties, unidirectional orientation in the liposomal membrane is required. Previous work of other groups, where MPs have been reconstituted and studied, did not include a final and universal approach (Rigaud JL, Levy D. (2003) Methods Enzymol. 372:65-86.; Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96.) and procedures have to be individually optimized for an enrichment of enzyme orientation. In most cases, however, orientation cannot be influenced and is thought to depend on the three-dimensional structure of the protein (Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96; Nordlund G et al. (2014) Nat. Commun. 5:4303.). We are currently developing and establishing a universal method to force unidirectional reconstitution of MPs by the aid of a molecular unit that can be attached to every protein. Our method is based on the observation that the F1F0 ATP synthase with its large hydrophilic head piece (F1 part) orients highly uniformly when embedded in preformed vesicles. We try to mimic this situation with a bioengineering approach that should be applicable to a wide variety of proteins. Once such a method is fully established, the interplay of two or more membrane proteins can be investigated more quantitatively.

The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap.

F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

How the Nucleus and Mitochondria Communicate in Energy Production During Stress: Nuclear MtATP6, an Early-Stress Responsive Gene, Regulates the Mitochondrial F1F0-ATP Synthase Complex

A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

Modelling the influence of thermal effects induced by radio frequency electric field on the dynamics of the ATPase nano-biomolecular motors

We model the dynamics of the F0 component of the F0F1-ATPase mitochondrion-based nano-motor operating in a stochastically-fluctuating medium that represents the intracellular environment. The stochastic dynamics are modeled via Langevin equation of motion wherein fluctuations are treated as white noise. We have investigated the influence of an applied alternating electric field on the rotary motion of the F0 rotor in such an environment. The exposure to the field induces a temperature rise in the mitochondrion’s membrane, within which the F0 is embedded. The external field also induces an electric potential that promotes a change in the mitochondrion’s transmembrane potential (TMP). Both the induced temperature and the change in TMP contribute to a change in the dynamics of the F0. We have found that for external fields in the radio frequency (RF) range, normally present in the environment and encountered by biological systems, the contribution of the induced thermal effects, relative to that of the induced TMP, to the dynamics of the F0 is more significant. The changes in the dynamics of the F0 part affect the frequency of the rotary motion of the F0F1-ATPase protein motor which, in turn, affects the production rate of the ATP molecules.

Unravelling the Symmetry Mismatch of the Two Coupled Rotary Motors of a Single FOF1-ATP Synthase by Three Color Fret

FoF1-ATP synthase is the enzyme that provides the ‘chemical energy currency’ adenosine triphosphate, ATP, for living cells. The enzyme consists of the membrane-embedded Fo part and the F1 part with three catalytic nucleotide binding sites. The formation of ATP is accomplished by a stepwise internal rotation of subunits within both parts. During ATP synthesis, proton translocation through the Fo part drives a 10-step rotary motion of the c10 ring with respect to the non-rotating subunits a and b2 [1]. This rotation is transmitted to the γ and ε subunits of the F1 part which perform three 120° steps per full rotation [2]. To localize transient elastic energy storage within the enzyme due to the symmetry mismatch, we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme. Cy5 is attached to one c subunit and EGFP to the a subunit of the Fo part [3]. The F1 part has a fluorophore bound at the ε subunit. To reduce photophysical artifacts due to spectral fluctuations of the fluorophores, a duty cycle-optimized alternating three laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors in a single enzyme revealed elastic deformations in the rotor parts of Fo and F1 during catalysis. [1] M. G. Düser, N. Zarrabi, D. J. Cipriano, S. Ernst, G. D.Glick, S.D. Dunn, M. Börsch (2009) EMBO Journal 28, 2689-2696. [2] B. Zimmermann, M. Diez, N. Zarrabi, P. Gräber, M. Börsch (2005) EMBO Journal 24, 2053-2063. [3] M. G. Düser, Y. Bi, N. Zarrabi, S.D. Dunn, M. Börsch (2008) J. Biol. Chem. 283, 33602-33610.

Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 subunit

F1Fo-ATP synthase is a key enzyme of mitochondrial energy provision producing most of cellular ATP. So far, mitochondrial diseases caused by isolated disorders of the ATP synthase have been shown to result from mutations in mtDNA genes for the subunits ATP6 and ATP8 or in nuclear genes encoding the biogenesis factors TMEM70 and ATPAF2. Here, we describe a patient with a homozygous p.Tyr12Cys mutation in the epsilon subunit encoded by the nuclear gene ATP5E. The 22-year-old woman presented with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, mild mental retardation and developed peripheral neuropathy. Patient fibroblasts showed 60-70% decrease in both oligomycin-sensitive ATPase activity and mitochondrial ATP synthesis. The mitochondrial content of the ATP synthase complex was equally reduced, but its size was normal and it contained the mutated epsilon subunit. A similar reduction was found in all investigated F1 and Fo subunits with the exception of Fo subunit c, which was found to accumulate in a detergent-insoluble form. This is the first case of a mitochondrial disease due to a mutation in a nuclear encoded structural subunit of the ATP synthase. Our results indicate an essential role of the epsilon subunit in the biosynthesis and assembly of the F1 part of the ATP synthase. Furthermore, the epsilon subunit seems to be involved in the incorporation of subunit c to the rotor structure of the mammalian enzyme.

Conserved Negative Charges in the Transmembrane Segments of Subunit K of the NADH:Ubiquinone Oxidoreductase Determine Its Dependence on YidC for Membrane Insertion

All members of the Oxa1/Alb3/YidC family have been implicated in the biogenesis of respiratory and energy transducing proteins. In Escherichia coli, YidC functions together with and independently of the Sec system. Although the range of proteins shown to be dependent on YidC continues to increase, the exact role of YidC in insertion remains enigmatic. Here we show that YidC is essential for the insertion of subunit K of the NADH:ubiquinone oxidoreductase and that the dependence is due to the presence of two conserved glutamate residues in the transmembrane segments of subunit K. The results suggest a model in which YidC serves as a membrane chaperone for the insertion of the less hydrophobic, negatively charged transmembrane segments of NuoK.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

Enhancement of the hydrolysis activity of F0F1‐ATPases using 60 Hz magnetic fields

The effects of extremely low frequency (ELF) magnetic fields on membrane F(0)F(1)-ATPase activity have been studied. When the F(0)F(1)-ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F(0)F(1)-ATPase was inhibited by N,N-dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F-DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F(0)F(1)-ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F(0)F(1)-ATPase may be an end-point affected by magnetic fields.

Erucylphosphohomocholine-induced apoptosis in human glioma cells: role of the oligomycin-sensitive F0 part of mitochondrial H+-ATP-synthase

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.

Δψ and ΔpH are equivalent driving forces for proton transport through isolated F0 complexes of ATP synthases

The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F1F0 holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H+-transport through F0. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H+/(s×F0) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H+-transport at initial rates of 6300 H+/(s×F0) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.

SNP mapping of QTL affecting growth and fatness on chicken GGA1

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351–353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.

SNP mapping of QTL affecting growth and fatness on chicken GGA1

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351–353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.

Biological effects of deuteronation: ATP synthase as an example

BackgroundIn nature, deuterium/hydrogen ratio is ~1/6600, therefore one of ~3300 water (H2O) molecules is deuterated (HOD + D2O). In body fluids the ratio of deuterons to protons is ~1/15000 because of the lower ionization constant of heavy water. The probability of deuteronation rather than protonation of Asp 61 on the subunit c of F0 part of ATP synthase is also ~1/15000. The contribution of deuteronation to the pKa of Asp 61 is 0.35.Theory and DiscussionIn mitochondria, the release of a deuteron into the matrix side half-channel of F0 is likely to be slower than that of a proton. As another example, deuteronation may slow down electron transfer in the electron transport chain (ETC) by interfering with proton coupled electron transport reactions (PCET), and increase free radical production through the leakage of temporarily accumulated electrons at the downstream complexes.ConclusionDeuteronation, as exemplified by ATP synthase and the ETC, may interfere with the conformations and functions of many macromolecules and contribute to some pathologies like heavy water toxicity and aging.

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.

Quel rôle spécifique pour ANT2 dans une cellule cancéreuse ?

In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Three isoforms with different kinetic properties are encoded from three different genes in Human: the muscle specific ANT1 and the ubiquitary ANT3 isoforms export ATP produced by mitochondrial oxidative phosphorylation (OXPHOS). The ANT2 isoform is specifically expressed in proliferative cells with a predominant glycolytic metabolism and is associated with cellular undifferentiation which is a major characteristic in carcinogenesis. Its role would be to import into mitochondria ATP produced by the glycolysis, energy essential to several intramitochondrial functions, particularly to maintenance of the membrane potential (Delta Psi m), conditioning cellular survival and proliferation. The mechanism of regeneration of this Delta Psi m gradient would involve at least three major proteins: the hexokinase II isoform, the ANT2 isoform and the F1 part of the mitochondrial ATP synthase complex. Taking into account this major role of ANT2 in cell proliferation and the very low expression of this isoform in differentiated tissues, this protein or its transcript could be chosen as a target for an anticancer strategy. Furthermore, previous studies showed that molecules of the cisplatin family, used as chemotherapeutic agents, led to the destruction of the mitochondrial membrane potential and thus to cell death. Does the anticancer effect of these molecules result, at least partially, from this mitochondrial aggression? If it is the case, the ANT2 isoform, mainly involved in the generation of this potential by its ATP4-/ADP3- exchange, could be considered as a more specific targeting by an RNA interference approach.