F<Subscript>1</Subscript>-ATPase as an auto-oscillatory system

In a relatively simple mathematical model we analyze the possibility of auto-oscillatory modes of F₁-ATPase during ATP hydrolysis.     

Mitochondrial and cell-surface F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase in innate and acquired cardioprotection

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F₀F₁ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F₀F₁ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F₀F₁ATPsynthase regulation by the inhibitory protein IF₁ in heart preconditioning strategies; ii) the structure and function of mitochondrial F₀F₁ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F₀F₁ ATP synthase in search for possible actors of its regulation, such as IF₁ and calmodulin, at cell surface.     

Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

Protein profiling in F<Subscript>1</Subscript> and F<Subscript>2</Subscript> generations of two tomato genotypes differing in ripening time

Pericarp polypeptide profiles were analyzed at three ripening stages in the F₁ hybrid and the F₂ population from the cross between the accessions: LA1385 (Lycopersicon esculentum var. cerasiforme) and 804627 (L. esculentum, a homozygous genotype for the nor mutant). Six polymorphic polypeptides were observed in LA1385, while no polymorphic polypeptides among ripening stages was observed in 804627. On the other hand, some polypeptides in the F₁ hybrid were not observed in the parents whereas others were present in both parental genotypes and were unnoticeable in the hybrid genotype. From a cluster analysis on the protein profiles of the F₂ population, the differential expression of proteins allowed to distinguish mature green (MG) stage from the others two stages, while for breaker stage (BR) and red ripe stage, the genetic background was more important in forming groups. The differential expression of proteins could be associated with fruit morphology traits such as a 72 kDa polypeptide present in MG stage with fruit diameter, height and mass and a 47 kDa polypeptide found in BR with fruit shelf life.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Zooming in on ATP Hydrolysis in F<Subscript>1</Subscript>

We summarize our current view of the reaction mechanism in F₁-ATPase as it has emerged from experiment, theory, and computational studies over the last several years. ATP catalysis in the catalytic binding pockets of F₁ takes place without the release of any significant free energy and is efficiently driven by the combined action of two water molecules utilizing a so-called protein-relay mechanism. The chemical reaction itself is controlled by the spatial position of a key arginine residue.     

A rapidly new-typed detection of norovirus based on F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATPase molecular motor biosensor

In order to adapt port rapid detection of food borne norovirus, presently we developed a new typed detection method based on F₀F₁-ATPase molecular motor biosensor. A specific probe was encompassed the conservative region of norovirus and F₀F₁-ATPase within chromatophore was constructed as a molecular motor biosensor through the “ε-subunit antibody-streptomycin-biotin-probe” system. Norovirus was captured based on probe-RNA specific binding. Our results demonstrated that the Limit of Quantification (LOQ) is 0.005 ng/mL for NV RNA and also demonstrated that this method possesses specificity and none cross-reaction for food borne virus. What’s more, the experiment used this method could be accomplished in 1 h. We detected 10 samples by using this method and the results were consistent with RT-PCR results. Overall, based on F₀F₁-ATPase molecular motors biosensor system we firstly established a new typed detection method for norovirus detection and demonstrated that this method is sensitive and specific and can be used in the rapid detection for food borne virus.     

Effect of Prostaglandins E<Subscript>1</Subscript> and F<Subscript>1α</Subscript> on Biosynthesis of Collagen

THE prostaglandins (PG) are possible mediators of inflammation. Prostaglandins E and F are present in inflammatory exudates^1–3 and could be related to the increase of collagen biosynthesis associated with inflammation. Vane and his colleagues^4–6 recently observed that indomethacin, aspirin and sodium salicylate potently block the biosynthesis of prostaglandins. These anti-inflammatory drugs are also inhibitors of collagen biosynthesis^7,8. Morphological studies⁹ have revealed increased deposition of collagen or collagen-related elements in organ cultures of chick embryo skin containing prostaglandins E₁ and B₁. We report here results which indicate stimulation of collagen biosynthesis by prostaglandins E₁ and F_1α evaluated by hydroxylation of proline and lysine and glycosylation of hydroxylysine in 10 day chick embryo tibiae.     

The ectopic F<Subscript>O</Subscript>F<Subscript>1</Subscript> ATP synthase of rat liver is modulated in acute cholestasis by the inhibitor protein IF<Subscript>1</Subscript>

Rat liver plasma membranes contain F_OF₁ complexes (ecto-F_OF₁) displaying a similar molecular weight to the mitochondrial F_OF₁ ATP synthase, as evidenced by Blue Native PAGE. Their ATPase activity was stably reduced in short-term extra-hepatic cholestasis. Immunoblotting and immunoprecipitation analyses demonstrated that the reduction in activity was not due to a decreased expression of ecto-F_OF₁ complexes, but to an increased level of an inhibitory protein, ecto-IF₁, bound to ecto-F_OF₁. Since cholestasis down regulates the hepatic uptake of HDL-cholesterol, and ecto-F_OF₁ has been shown to mediate SR-BI-independent hepatic uptake of HDL-cholesterol, these findings provide support to the hypothesis that ecto-F_OF₁ contributes to the fine control of reverse cholesterol transport, in parallel with SR-BI. No activity change of the mitochondrial F_OF₁ ATP synthase (m-F_OF₁), or any variation of its association with m-IF₁ was observed in cholestasis, indicating that ecto-IF₁ expression level is modulated independently from that of ecto-F_OF₁, m-IF₁ and m-F_OF₁.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

SSR-based genetic linkage map construction in pistachio using an interspecific F<Subscript>1</Subscript> population and QTL analysis for leaf and shoot traits

Pistachio is one of the most commercially important nut trees in the world. To characterize the genetic controls of horticultural traits and facilitate marker-assisted breeding in pistachio, we constructed an SSR-based linkage map using an interspecific F₁ population derived from a cross between the cultivar “Siirt” (Pistacia vera L.) and the monoecious Pa-18 genotype of Pistacia atlantica Desf. This population was also used for the first QTL analysis in pistachio on leaf and shoot characters. In total, 1312 SSR primers were screened, and 388 loci were successfully integrated into parental linkage maps. The Siirt maternal map contained 306 markers, while the “Pa-18” paternal map included 285 markers along the 15 linkage groups. The Siirt map spanned 1410.4 cM, with an average marker distance of 4.6 cM; the Pa-18 map covered 1362.5 cM with an average marker distance of 4.8 cM. Phenotypic data were collected during the growing seasons of 2015 and 2016 for four traits: leaf length (LL), leaf width (LW), leaf length/leaf width ratio (LWR), number of leaflet pairs (NLL), and young shoot color (YSC). A total of 17 QTLs were identified in the parental maps. Four QTLs for LL and LW were located on LG2 and LG4, while four QTLs for LWR ratio on LG13 and LG14, two QTLs for NLL and two QTLs for YSC were on LG7 and LG9, respectively, with similar positions in both parental maps. The SSR markers, linkage maps, and QTLs reported here will provide a valuable resource for future molecular and genetic studies in pistachio.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

3,5-Diiodo-L-Thyronine increases F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats

Short-term effects of 3,5-L-diiodothyronine (T₂) administration to hypothyroid rats on F_oF₁-ATP synthase activity were investigated in liver mitochondria. One hour after T₂ injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F_oF₁-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T₂ administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F_oF₁-ATP synthase was found, ruling out a T₂ genomic effect. In T₂-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T₂ administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T₂-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F_oF₁-ATP synthase activity observed following injection of T₂ to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.     

The F<Subscript>O</Subscript>F<Subscript>1</Subscript> ATP synthase: from atomistic three-dimensional structure to the rotary-chemical function

There are numerous studies describing how growth conditions influence the efficiency of C₄ photosynthesis. However, it remains unclear how changes in the biochemical capacity versus leaf anatomy drives this acclimation. Therefore, the aim of this study was to determine how growth light and nitrogen availability influence leaf anatomy, biochemistry and the efficiency of the CO₂ concentrating mechanism in Miscanthus × giganteus. There was an increase in the mesophyll cell wall surface area but not cell well thickness in the high-light (HL) compared to the low-light (LL) grown plants suggesting a higher mesophyll conductance in the HL plants, which also had greater photosynthetic capacity. Additionally, the HL plants had greater surface area and thickness of bundle-sheath cell walls compared to LL plants, suggesting limited differences in bundle-sheath CO₂ conductance because the increased area was offset by thicker cell walls. The gas exchange estimates of phosphoenolpyruvate carboxylase (PEPc) activity were significantly less than the in vitro PEPc activity, suggesting limited substrate availability in the leaf due to low mesophyll CO₂ conductance. Finally, leakiness was similar across all growth conditions and generally did not change under the different measurement light conditions. However, differences in the stable isotope composition of leaf material did not correlate with leakiness indicating that dry matter isotope measurements are not a good proxy for leakiness. Taken together, these data suggest that the CO₂ concentrating mechanism in Miscanthus is robust under low-light and limited nitrogen growth conditions, and that the observed changes in leaf anatomy and biochemistry likely help to maintain this efficiency.     

The Effects of Copper (II) Ions on <Emphasis Type="Italic">Enterococcus hirae</Emphasis> Cell Growth and the Proton-Translocating F<Subscript>o</Subscript>F<Subscript>1</Subscript> ATPase Activity

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (~+35 mV) to negative ones (~−220 mV). An oxidizer copper (II) ions (Cu²⁺) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent β subunit of F₁ of the F_oF₁ ATPase) both. Also ATPase activity and proton–potassium ions exchange were assessed with and without N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. In both cases (DCCD ±), even low Cu²⁺ concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu²⁺ markedly decreased the number of SH-groups in the presence of K⁺ ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu²⁺ blocked ATP-installed increase in SH-groups number in ATCC9790. H⁺–K⁺-exchange of bacteria was markedly inhibited by Cu²⁺, but stronger effects were detected together with DCCD. Moreover, discrimination between Cu²⁺ and other bivalent cation—Ni²⁺ was shown. It is suggested that Cu²⁺ ions inhibit E. hirae cell growth by direct affect on the F_oF₁ ATPase leading to conformational changes in this protein complex and decrease in its activity.     

Identification of a Conserved Calmodulin-Binding Motif &;#x220A; the Sequence of F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase Inhibitor Protein

The natural inhibitor proteins IF₁ regulate mitochondrial F₀F₁ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF₁, two bovine IF₁ synthetic peptides, as well as two homologous proteins from yeast, namely IF₁ and STF₁. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF₁ interaction is Ca²⁺-dependent. Bovine and yeast IF₁ have intermediate binding affinity for CaM, while the K_d (dissociation constant) of the STF₁-CaM interaction is slightly higher. Binding studies of CaM with bovine IF₁ synthetic peptides allowed us to identify bovine IF₁ sequence 33–42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF₁ and STF₁ sequences. In addition, the same region in bovine IF₁ has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF₁ but not in STF₁. Based on the pH and Ca²⁺ dependence of IF₁ interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF₁ trafficking or additional IF₁ roles not yet clarified.     

Asymmetries,heterosis, and phenotypic profiles of red junglefowl,White Plymouth Rocks,and F<Subscript>1</Subscript> and F<Subscript>2</Subscript> reciprocal crosses

During the domestication of farm animals, humans have manipulated genetic variation for growth and reproduction through artificial selection. Here, data are presented for growth, reproductive, and behavior traits for the red junglefowl, a line of White Plymouth Rock chickens, and their F₁ and F₂ reciprocal crosses. Intra- and intergenerational comparisons for growth related traits reflected considerable additive genetic variation. In contrast, those traits associated with reproduction exhibited heterosis. The role of sexual selection was seen in the evolution of prominent secondary sexual ornaments that lend to female choice and male-male competition. The large differences between parental lines in fearfulness to humans were only mitigated slightly in the intercross generations. Whereas, overall F₁ generation heterosis was not transferred to the F₂, there was developmental stability in the F₂, as measured by relative asymmetry of bilateral traits. Through multigenerational analyses between the red junglefowl and the domestic White Plymouth Rocks, we observed plasticity and considerable residual genetic variation. These factors likely facilitated the adaptability of the chicken to a broad range of husbandry practices throughout the world.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.