Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.     

ATP synthase from Saccharomyces cerevisiae: location of the OSCP subunit in the peripheral stalk region

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase-avidin complex, the bound avidin has been localised close to the F(1) domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F(1) domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F(1) domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of alpha-subunits, which are on top of the F(1) domain distal from the F(o) membrane domain, the OSCP extends almost 10nm along the surface of F(1) down towards F(o) where it interacts with the C terminus of the b subunit, which extends up from F(o). The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S.cerevisiae. The map reveals a marked asymmetry in the F(o) part of the complex that can be attributed to subunits in the F(o) domain.     

Modular assembly of yeast mitochondrial ATP synthase

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.     

Solution structure of subunit F(6) from the peripheral stalk region of ATP synthase from bovine heart mitochondria

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.     

HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion

Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice.     

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.     

The rotary mechanism of ATP synthase

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.     

In vivo inhibition of the mitochondrial H+‐ATP synthase in neurons promotes metabolic preconditioning

A key transducer in energy conservation and signaling cell death is the mitochondrial H⁺‐ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H⁺‐ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H⁺‐ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid‐induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl‐xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H⁺‐ATP synthase as a target to prevent neuronal cell death.     

酵母线粒体ATP合酶生物合成及组装机制研究进展

线粒体ATP合酶是线粒体氧化磷酸化的关键酶,其功能缺陷会导致能量代谢障碍相关的线粒体疾病。线粒体ATP合酶是由多个亚基组成的蛋白复合物,其生物合成和组装是个复杂的生物过程。酵母是研究线粒体ATP合酶结构、生物合成和组装机制的模式实验材料之一,且相关研究取得了很多进展。本文概述了国内外用酿酒酵母研究线粒体ATP合酶的结构、调控线粒体ATP合酶亚基生物合成和组装的辅助蛋白及合酶的模块化组装过程的研究进展,以期为线粒体ATP合酶的工作机制及相关线粒体疾病的研究提供理论借鉴和参考依据。     

The peripheral stalk of the mitochondrial ATP synthase

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.     

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H⁺ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca²⁺‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca²⁺ binding site and the mechanism(s) through which Ca²⁺ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca²⁺‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca²⁺‐ATP hydrolysis, confer resistance to Ca²⁺‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

Binding affinities and protein ligand complex geometries of nucleotides at the F(1) part of the mitochondrial ATP synthase obtained by ligand docking calculations

F₀F₁ ATP synthases utilize a transmembrane electrochemical potential difference to synthesize ATP from ADP and phosphate. In this work, the binding modes of ADP, ATP and ATP analogues to the catalytic sites of the F₁ part of the mitochondrial ATP synthase were investigated with ligand docking calculations. Binding geometries of ATP and ADP at the three catalytic sites agree with X-ray crystal data; their binding free energies suggest an assignment to the ‘tight’, ‘open’ and ‘loose’ states. The rates of multi-site hydrolysis for two fluorescent ATP derivatives were measured using a fluorescence assay. Reduced hydrolysis rates compared to ATP can be explained by the ligand docking calculations.     

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.     

Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8

Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide–resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane‐embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt α‐helical secondary structure is evidenced by circular dichroism spectroscopy. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

Thermodynamic analysis of F1‐ATPase rotary catalysis using high‐speed imaging

F₁-ATPase (F₁) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F₁ rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F₁ catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F₁ rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (P_i). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P_i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG^‡ = 53–56 kJ mol⁻¹, the contributions of the enthalpy (ΔH^‡), and entropy (ΔS^‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH^‡ and ΔS^‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.