36° step size of proton‐driven c‐ring rotation in FoF1‐ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by F_oF₁‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the F_o motor powers catalysis in the F₁ motor. Although F₁ uses 120° stepping during ATP synthesis, models of F_o predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.     

The Structural and Functional Connection between the Catalytic and Proton Translocating Sectors of the Mitochondrial F 1 F 0 -ATP Synthase

The structural and functional connection between the peripheral catalytic F₁ sector and theproton-translocating membrane sector F₀ of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F₀I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F₁ to F₀ and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF₁) binds at one sideof the F₁F₀ connection. The carboxy-terminal segment of IF₁ apparently binds to OSCP. The42L-58K segment of IF₁, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F₁, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.     

Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C‐ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F₁F_O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F₁F_O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F₁F_O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F₁F_O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F₁F_O ATP synthase dimers. Destabilizing F₁F_O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F₁F_O ATP synthase dimers and involves the C‐ring.     

The unique histidine in OSCP subunit of F‐ATP synthase mediates inhibition of the permeability transition pore by acidic pH

The permeability transition pore (PTP) is a Ca²⁺‐dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H⁺ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F₁F_O (F)‐ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP‐dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch‐clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H⁺ are mediated by the F‐ATP synthase.     

Structural and functional characterization of FoF1-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_oF₁-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 F₁ (α/β and γ) and F_o (F_oI-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 IF₁ 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 F₁ and F_o-targeting inhibitors. We hypothesise that ecto-F_oF₁-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.     

IF<Subscript>1</Subscript> distribution in HepG2 cells in relation to ecto–F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase and calmodulin

F₀F₁ATPsynthase is now known to be expressed as a plasma membrane receptor for several extracellular ligands. On hepatocytes, ecto–F₀F₁ATPsynthase binds apoA–I and triggers HDL endocytosis concomitant with ATP hydrolysis. Considering that inhibitor protein IF₁ was shown to regulate the hydrolytic activity of ecto–F₀F₁ATPsynthase and to interact with calmodulin (CaM) in vitro, we investigated the subcellular distributions of IF₁, calmodulin (CaM), OSCP and β subunits of F₀F₁ATPsynthase in HepG2 cells. Using immunofluorescence and Western blotting, we found that around 50% of total cellular IF₁ is localized outside mitochondria, a relevant amount of which is associated to the plasma membrane where we also found Ca²⁺–CaM, OSCP and β. Confocal microscopy showed that IF₁ colocalized with Ca²⁺–CaM on plasma membrane but not in mitochondria, suggesting that Ca²⁺–CaM may modulate the cell surface availability of IF₁ and thus its ability to inhibit ATP hydrolysis by ecto–F₀F₁ATPsynthase. These observations support a hypothesis that the IF₁–Ca²⁺–CaM complex, forming on plasma membrane, functions in the cellular regulation of HDL endocytosis by hepatocytes.     

Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (P_i) to pyrophosphate (PP_i) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both P_i and PP_i, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. J. Cell. Physiol. 230: 3049–3056, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1

When mitochondrial respiration is compromised, the F₁F_o-ATP synthase reverses and consumes ATP, serving to maintain the mitochondrial membrane potential (Δψ_m). This process is mitigated by IF₁. As little is known of the cell biology of IF₁, we have investigated the functional consequences of varying IF₁ expression. We report that, (1) during inhibition of respiration, IF₁ conserves ATP at the expense of Δψ_m; (2) overexpression of IF₁ is protective against ischemic injury; (3) relative IF₁ expression level varies between tissues and cell types and dictates the response to inhibition of mitochondrial respiration; (4) the density of mitochondrial cristae is increased by IF₁ overexpression and decreased by IF₁ suppression; and (5) IF₁ overexpression increases the formation of dimeric ATP synthase complexes and increases F₁F_o-ATP synthase activity. Thus, IF₁ regulates mitochondrial function and structure under both physiological and pathological conditions.     

In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart

A method has been developed to allow the level of F₀F₁ATP synthase capacity and the quantity of IF₁ 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 IF₁ content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF₁ 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 IF₁ content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF₁. In addition, both in vivo and in vitro, 1.3-1.4 mol of IF₁ was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF₁ in the F₀ sector.     

Control of rotation of the F<Subscript>1</Subscript>F<Subscript>O</Subscript>-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF<Subscript>1</Subscript> 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 F₁F_O-ATPase activity by blocking γ subunit rotation at the α_DP/β_DP/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 α_DP/β_DP/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 α_DP/β_DP/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 F₁F_O-ATPase rotation, without affecting the CW-F₁F_O-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.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Unidirectional regulation of the F1FO-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF₁ subunits, prevent the wasteful CCW F₁F_O-ATPase activity by blocking γ rotation at the α_DP/β_DP/γ interface of the F₁ portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F₁-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF₁, it also binds to the α_DP/β_DP/γ interface of the F₁ of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α_DP/β_DP/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F₁F_O-ATPase rotation without affecting the CW-F₁F_O-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdF₁F_O-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF₁ but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF₁) of this unique ATP synthase nanomotor, essential for life.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

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.     

Shear stress‐mediated F1/FO ATP synthase‐dependent CO2 gas excretion from human pulmonary arteriolar endothelial cells

We studied the physiological role of flow through pulmonary arterioles in CO₂ gas exchange. We established human pulmonary arteriolar endothelial cells (HPAoEC). The cells demonstrated marked immunocytochemical staining of PECAM‐1, VEGF R2, ACE‐1, and CA type IV on their cell surface. Ten seconds shear stress stimulation caused the co‐release of H⁺ and ATP via the activation of F₁/F_O ATP synthase on the HPAoEC. F₁/F_O ATP synthase was immunocytochemically observed on the cell surface of non‐permeabilized HPAoEC. In the shear stress‐loaded HPAoEC culture media supernatant, ATPase activity increased in a time‐dependent manner. The HPAoEC were strongly stained for NTPDase 1, which partially co‐localized with purinergic P2Y1. The purinergic P2Y1 receptor agonist UTP (10^?6 M) significantly potentiated the shear stress‐induced increase in ATPase activity in the culture medium supernatant. Ten seconds shear stress stimulation also produced stress strength‐dependent CO₂ gas excretion from the HPAoEC, which was significantly reduced by the inhibition of F₁/F_O ATP synthase or CA IV on the endothelial cell (EC) surface. In conclusion, we have proposed a new concept of CO₂ exchange in the human lung, flow‐mediated F₁/F_O ATP synthase‐dependent H⁺ secretion, resulting in the facilitation of a dehydration reaction involving in plasma and the excretion of CO₂ gas from arteriolar ECs. J. Cell. Physiol. 227: 2059–2068, 2012. © 2011 Wiley Periodicals, Inc.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

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.     

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.     

Cloning of the cDNA for the Human ATP Synthase OSCP Subunit (ATP50) by Exon Trapping and Mapping to Chromosome 21q22.1-q22.2

Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP50 gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. (Nature 359:380, 1992) YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP50 subunit is a key structural component of the stalk of the mitochondrial respiratory chain F₁F₀-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21).