The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Caenorhabditis elegans MAI-1 protein, which is similar to mitochondrial ATPase inhibitor (IF1), can inhibit yeast F0F1-ATPase but cannot be transported to yeast mitochondria

In Caenorhabditis elegans, two proteins that are similar to mitochondrial ATPase inhibitor protein (IF₁) have been found and named MAI-1 and MAI-2. In this study, we overexpressed and purified both the proteins and examined their properties. Circular dichroism spectra indicated that both the MAI-1 and MAI-2 predominantly consisted of β- and random structure, and in contrast to mammalian IF₁, α-helixes were barely detected. Both MAI-1 and MAI-2 could inhibit yeast F₀F₁-ATPase, but the inhibition by MAI-1 was pH-independent. MAI-2-GFP fusion protein was transported to yeast mitochondria, but MAI-1-GFP was not. These results indicate that the MAI-2 is _{C. elegans} IF₁. MAI-1 seems to be a cytosolic protein and may regulate cytosolic ATPase(s).     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Mutagenesis Studies of the F1F0 ATP Synthase b Subunit Membrane Domain

A homodimer of b subunits constitutes the peripheral stalk linking the F₁ and F₀ sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F₁F₀ ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F₁F₀ ATPase and no detectable ATP-driven proton pumping activity. Expression of the b _N2A,T6A,Q10A subunit was also oxidative phosphorylation deficient, but the b _N2A,T6A,Q10A protein was incorporated into an F₁F₀ complex. Single amino acid substitutions had minimal reductions in F₁F₀ ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F₀, and that these interactions are strongest on the periplasmic side of the bilayer.     

Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside

Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.     

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.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

Mutagenic Analysis of the F 0 Stator Subunits

The a and b subunits constitute the stator elements in the F₀ sector of F₁F₀-ATP synthase.Both subunits have been difficult to study by physical means, so most of the information onstructure and function relationships in the a and b subunits has been obtained using mutagenesisin combination with biochemical methods. These approaches were used to demonstrate thatthe a subunit in association with the ring of c subunits houses the proton channel throughF₁F₀-ATP synthase. The map of the amino acids contributing to the proton channel is probablycomplete. The two b subunits dimerize, forming an extended flexible unit in the peripheralstalk linking the F₁ and F₀ sectors. The unique characteristics of specific amino acid substitutionsaffecting the a and b subunits suggested differential effects on rotation during F₁F₀-ATPaseactivity.     

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

Monoclonal and polyclonal antibodies directed against peptides of F₁-ATPase or F₁F₀-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F₁F₀-antibody complexes of various masses, seems to exclude the functional rotation of F₁ around F₀ during catalysis.Abbreviations IF₁ natural protein inhibitor of the ATPase-ATP synthase - OSCP oligomycin sensitivity-conferring protein - DCCD dicyclohexylcarbodiimide - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoreses - F₁ F₁-ATPase, coupling factor F₁ of ATPase - F₁F₀ F₁F₀-ATP synthase, ATPase-ATP synthase complex     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Construction and assessment of reaction models between F1F0-synthase and organotin compounds: molecular docking and quantum calculations

Organotin compounds are the active components of some fungicides, which are potential inhibitors of the F₁F₀-ATP synthase. The studies about the reaction mechanism might indicate a pathway to understand how these compounds work in biological systems, however, has not been clarified so far. In this line, molecular modeling studies and density functional theory calculations were performed in order to understand the molecular behavior of those compounds when they interact with the active site of the enzyme. Our findings indicate that a strong interaction with His132 can favor a chemical reaction with organotin compounds due to π–π stacking interactions with aromatic rings of organotin compounds. Furthermore, dependence on molecule size is related to possibility of reaction with the amino acid residue His132. Thus, it can also be noticed, for organotin compounds, that substituents with four carbons work by blocking the subunit a, in view of the high energy transition found characterized by steric hindrance.     

Atrazine binds to F1F0-ATP synthase and inhibits mitochondrial function in sperm

Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F₁F₀-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F₁F₀-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F₁F₀-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F₁F₀-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology.     

Hypothyroidism Leads to a Decreased Expression of Mitochondrial F0F1-ATP Synthase in Rat Liver

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F₀F₁-ATP synthase are accompanied by a decrease in the amount of immunodetected -F₁, F₀1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F₁ subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F₀F₁-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F₀F₁-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.     

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.     

Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F₀F₁-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F₀ to the endergonic conformational changes in F₁ required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the subunit of F₁ rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F₁ and membrane-bound F₀F₁. Furthermore, the inhibition of this rotation by the modification of F₀ with DCCD suggests that rotation in F₁ is obligatorily coupled to rotation in F₀ as an integral part of the coupling mechanism.     

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α_DP- and β_DP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β_E-subunits. The nucleotide binding site in β_E-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.