Subunit interaction in the mitochondrial H+-translocating ATPase. Association of the 26,500-dalton atpase binding protein and oligomycin sensitivity conferral protein with F1-ATPase

The rat liver 26,500-dalton ATPase binding protein and beef heart oligomycin sensitivity conferral protein are able to interact with the rat liver Type II ATPase to form discrete complexes. The equilibrium constants for these interactions are similar and each forms a 1:1 complex with the ATPase. The reassociated complex of Type II ATPase and 26,500-dalton ATPase binding protein or of oligomycin sensitivity conferral protein and Type II ATPase has properties similar to that of Type I ATPase. Dimerization of oligomycin sensitivity conferral protein by oxidation with copper phenanthroline chelate abolishes its ability to interact with the Type II ATPase. The isoelectric point and amino acid composition of the 26,500-dalton ATPase binding protein and oligomycin sensitivity conferral protein are similar. The polypeptide patterns produced by cyanogen bromide cleavage indicates a similar but nonidentical pattern to the 26,500-dalton ATPase binding protein and the oligomycin sensitivity conferral protein.     

Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide

Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide were studied in beef heart submitochondrial particles, and the results suggest that the two inhibitors have different binding sites with respect to the membrane and to F1. Oligomycin seems to be bound to a subunit or a part of a subunit in F0, which is localized superficially, and which is influenced by F1, since the presence of F1 considerably lowers the rate of inhibition. The oligomycin binding site further seems to be influenced by the different conformational states of F1 occurring during the catalytic cycle of the enzyme. The binding site of DCCD on F0, on the other hand, seems to be deeply embedded in the membrane and not influenced by F1.     

Discrimination between the binding sites of modulators of the H+-translocating ATPase activity in rat liver mitochondrial membranes

The properties of the components of the mitochondrial ATPase which interact with modulators of energy transduction have been examined. The chromatographic behavior and the size of the components which bind trialkyl tins, carbodiimides and uncouplers, have been shown to be different. However, they all appear to be proteolipids with apparent molecular weights around 10,000. On this basis it is proposed that these inhibitors act at different sites in the membrane sector of the ATP synthase of rat liver mitochondria.     

Carbodiimide-binding protein of H+-translocating ATPase and inhibition of H+ conduction by dicyclohexylcarbodiimide.

H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified F0 from a thermophilic bacterium PS3 (TF0), the following results were obtained. 1. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5 times 10(3) M(-1)-min(-1). 2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+. 3. By extracting TF0 with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6,000. 4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6,500. 5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.     

ATP synthase from bovine mitochondria: sequences of imported precursors of oligomycin sensitivity conferral protein, factor 6, and adenosinetriphosphatase inhibitor protein

Oligomycin sensitivity conferral protein (OSCP), factor 6 (F6), and ATPase inhibitor protein are all components of the ATP synthase complex of bovine mitochondria. They are encoded in nuclear DNA. Complementary DNA clones encoding the precursors of these proteins have been isolated from a bovine library by using mixtures of synthetic oligonucleotides as hybridization probes, and their DNA sequences have been determined. The deduced protein sequences show that the OSCP, F6, and inhibitor proteins have N-terminal presequences of 23, 32, and 25 amino acids, respectively. These presequences are not present in the mature proteins. It is assumed that they serve to direct the proteins into the mitochondrial matrix. The cDNA clones have also been employed as hybridization probes to investigate the genetic complexity of the three proteins in cows and humans. These experiments indicate that the bovine and human inhibitor and bovine F6 proteins are encoded by single genes but suggest the possibility of the presence in both species of more than one gene (or pseudogenes) for the OSCP.     

On the identification of the uncoupler binding protein of bovine mitochondrial oligomycin sensitive ATPase

The 55,000 dalton polypeptide component of the membrane sector of the mitochondrial oligomycin sensitive ATPase has been purified by recycling chromatography on BioGel P-100. The amino acid composition of the purified polypeptide differs significantly from that of the α-subunit of F₁ with which it shares a similar apparent molecular weight. However, the amino acid composition of the former is identical to that of the Factor B polypeptide, which is known to occur in oligomeric forms. Evidence is presented which suggests that the mitochondrial uncoupler binding proteins and the various oligomeric forms of the Factor B polypeptide, including the 55,000 dalton species described in the present report, are identical.     

On the functional role of coupling factor B in the mitochondrial H+ -ATPase

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56). FB was completely inactivated by 38 micron of copper o-phenanthroline or 0.63 mM iodosobenzoate, and the kinetics were consistent with intramolecular disulfide formation as were polyacrylamide gel patterns which showed that FB which had been treated with copper o-phenanthroline had a different mobility from that of untreated FB. ATP-Pi exchange activity and ATP-induced binding of bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol (oxonol VI) to H+ -ATPase were also inhibited by the thiol oxidizing reagents, although oligomycin-sensitive ATPase activity was unaffected. F0 isolated from H+ -ATPase rebinds purified F1 with the restoration of ATP-induced oxonol-binding activity. Prior treatment of F0 (but not of F1) with copper o-phenanthroline abolished the oxonol-binding activity of reconstituted F0-F1. 115Cd binds tightly to H+ -ATPase and the bound protein can be recovered by gel electrophoresis in phosphate buffer in the presence of sodium dodecyl sulfate at a position corresponding to FB. Prior treatment of the H+ -ATPase with copper o-phenanthroline abolished 115Cd binding. The results indicate that the major effect of these inhibitors is on FB dithiol and leave little doubt that Cd2+ is indeed bound to a vicinal dithiol group.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.     

Primary structure of the OSCP protein that confers sensitivity to oligomycin on the mitochondrial H+-ATPase complex. I. Tryptic and cyanogen bromide peptides

Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.     

Several classes of binding sites for metals and nucleotides on yeast mitochondrial oligomycin sensitive ATPase.

The binding properties of Mg2+, Mn2+ and Co2+ to yeast mitochondrial oligomycin sensitive ATPase complex are studied, as reflected by their catalytic effect (hydrolysis of ATP or pNPP, a pseudo substrate) or by a physical parameter (atomic absorption, electron paramagnetic reasonance of Mn2+, enhanced fluorescence of chelating chlorotetracyclin). At least two classes of sites with very different affinities respectively around 10(-5) M and 10(-4) M are demonstrated: high affinity sites for cations which participate in pNPP hydrolysis and can bind ADP or ATP, although they have a poor efficiency for ATP hydrolysis, and low affinity sites for cations which participate efficiently in both pNPP and ATP hydrolysis. The possibility that the tight site class has itself two sub-classes is also discussed.     

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.     

Subunit exchange and the role of dimer flexibility in DNA binding by the Fis protein

Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.     

Isolation of H+ -translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-beta-D-glucoside (n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H(+) pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine-fluorescence quenching was observed in the presence of ATP-Mg(2+), indicating the H(+) pumping. The H(+) pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO(3), an inhibitor specific to tonoplast-type (V-type) H(+)-ATPase.     

Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex

In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus.

Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.     

Lability of coupling between proton translocase and ATPase of mitochondrial H+-ATPase complex]

The interrelationship between the ATPase and H+-translocase functions of mitochondrial H+-ATPase was studied. The efficiency of the functioning was estimated by the value of coupling coefficient (Kc), which is represented by a ratio of proton translocation rate versus ATP coupling hydrolysis rate. It was shown that under conditions of increased concentrations of ATP and low concentrations of oligomycin the value of Kc is decreased. The increase in the concentration of valinomycin results in an increase of Kc. It was also found that the H+-ATPase activity shows a considerable increase during incubation of mitochondria, reaching its maximum with respect to both functions 1--2 min after addition of ATP. The data obtained are indicative of a lack of tight coupling between the H+-translocase and ATPase functions of mitochondrial H+-ATPase. The mechanism of action of H+-ATPase is discussed.