Oligomycin-sensitive ATPase Complex Research Articles

Structural Interactions of the Oligomycin Sensitivity-Conferring Protein in the Yeast ATP Synthase

The structure/function relationship of oligomycin sensitivity-conferring protein (OSCP), subunit 5 of the mitochondrial ATP synthase, from yeastSaccharomyces cerevisiaehas been studied by a combination of genetic and biochemical methods. OSCP was studied by deletion mutagenesis of the N- and C-terminal regions by modifying the gene coding for OSCP. Two deletion mutations were made immediately downstream of the leader peptide of OSCP and five were made at the C-terminus. OSCP was functional with deletions of amino acids 3 to 17 (ND15) and of the last 8 amino acids (CD8), while deletion of amino acids 3 to 31 (ND29) and the last 9 amino acids (CD9) inactivated the ATP synthase, as determined byin vivoanalysis. The deletion mutants were expressed inEscherichia coli,purified, and studied byin vitroreconstitution studies. Circular dichroism studies suggested that the mutant proteins, with the possible exception of ND29, were folded in a similar fashion as wild-type OSCP. Mutants ND15 and CD8 were able to reconstitute an oligomycin-sensitive ATPase complex, although not as well as wild-type OSCP, while ND29 and CD9 were completely ineffective. Binding studies of ND29 and CD9 indicate that these mutants in OSCP were unable to bind to the membrane portion of the ATP synthase, F0, and these results were supported by competition binding studies. These results support the hypothesis that the N- and C-terminal regions of subunit 5 interact with F0and suggest that the central region interacts with F1.

Myocardial acidosis and the mitigation of tissue ATP depletion in ischemic cardiac muscle: the role of the mitochondrial ATPase.

A number of reports have now appeared demonstrating a marked inhibition of the mitochondrial oligomycin-sensitive ATPase as the result of the interruption of blood flow (ischemia) in a number of tissues including liver,1,2 kidney,3,4 skeletal muscle5 and cardiac muscle.6–9 By and large, the ATPase inhibition observed in ischemic tissues appears to be reversible if blood flow is resumed early enough,2,4,6,9 suggesting that this inhibition represents a regulatory response of the oligomycin-sensitive ATPase complex to one or more metabolic alterations within the ischemic cells.

The pho1 mutation. A frameshift, and its compensation, producing altered forms of physiologically efficient ATPase in yeast mitochondria.

The pho1 mutation belongs to the OL12 gene on mitochondrial DNA of Saccharomyces cerevisiae, which codes for a membrane factor subunit of the mitochondrial ATPase (apparent molecular mass 20 kDa). We analysed the ATPase complex from the pho1 mutant and from three revertants, after immunoprecipitation from mitochondrial extracts, by dodecyl sulphate/acrylamide gel electrophoresis. In two revertants the OL12 gene product appeared as an abundant slower migrating peptide, while in the pho mutant, two bands appeared in very low amounts. For the third revertant, a strong band appeared at the normal level. Sequencing of the OL12 gene from these strains gave the following results: the pho1 mutation is a frameshift, arising by insertion of an extra thymidine into a group of three. Two of the revertants contain the same group of four thymidines, but genetic compensation of the frameshift occurs 24 base pairs downstream by the loss of four bases, implying a deficit of one codon. The third revertant has recovered the normal three-thymidine sequence. There is excellent correlation between the modified sequences and electrophoretic migration of the peptide product. Owing to the leakiness of the pho1 phenotype (reduced but not nil growth rate on oxidizable carbon sources, 5-10% highly oligomycin-sensitive ATPase complex, low amounts of OL12 gene product peptides), some translational correction of the frameshift is bound to occur. Based on these results, the compatibility of abnormal ATPase architecture with modified energetic efficiency is discussed.

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase.

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimyristoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18-28 degrees C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots of Km also show breaks in the same temperature interval, with the exception of the dioleoyl-enzyme vesicles, where Km is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24 degrees C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3-18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.

Amino acid composition of a new mitochondrially translated proteolipid isolated from yeast mitochondria and from the OSATPase complex

A new mitochondrially translated 10000 Mr proteolipid was isolated from yeast mitochondria. This proteolipid was purified by phosphocellulose chromatography, followed by reverse phase HPLC. This proteolipid was also extracted from the oligomycin sensitive ATPase complex and purified by HPLC. Its amino acid composition is different from the Dicyclohexylcarbodiimide binding protein.

The yeast mitochondrial adenosine triphosphatase complex. Subunit stoichiometry and physical characterization.

Immunoprecipitation of uniformly labeled yeast submitochondrial preparations using a subunit-specific or a holoenzyme antiserum has been employed to determine the subunit stoichiometry of the oligomycin-sensitive ATPase complex. The Triton-solubilized enzyme consists of 10 types of subunits. The number of copies of each subunit, in order of decreasing molecular weight, is 3:3:1:2:1:2:2:1:2:3. on the basis of the stoichiometry data, the ATPase complex has a molecular weight of 5.8 x 10(5) and contains a minimum of 20 polypeptide chains. Analysis of water-soluble ATPase (F1-ATPase) indicates that the stoichiometry of the three largest subunits of the enzyme is preserved in the absence of the other subunits. The molecular weights of both forms of the ATPase, derived from stoichiometry data, agree well with measurements obtained from gel filtration and sedimentation studies. The implications of these data for the structure, function, and assembly of the complex are discussed.

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the [formula omitted] carbodiimide-binding protein is synthesized outside the mitochondria

1. 1. Radioactive N,N′- dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N,N′- dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N′- dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. 2. Radioactive N,N′- dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N′- dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. 3. The results together indicate that the N,N′- dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.

Components and mechanism of action of ATP-driven proton pomps

We have studied the composition of ATP-driven proton pumps from bovine heart mitochondria and have reconstituted the oligomycin-sensitive ATPase complex from its individual components. The complex contains 9 to 10 subunits of which 5 are assembled in the soluble F1 protein, 2 are required for the attachment of F1 to the membrane and 2 form the proton channel within the membrane. With the help of information obtained from studies of the chloroplast and the bacterial proton pumps, we can tentatively assign a function to each of the subunits of the pump. The position of F1 outside of the membrane seen in electron micrographs of negatively stained preparations, does not appear to be an artifact. Evidence from immunological studies, chemical derivatizations as well as further electron microscopy (positive staining and freeze-etching), support this statement. We describe in this paper a 28 000-dalton polypeptide which has been isolated from the mitochondria membrane and is required for the reconstitution of oligomycin-sensitive ATPase and 32Pi-ATP exchange activity. We propose a mechanism of action of the proton pump in which the key energy-yielding reaction is the binding of Mg2+ to the protein. The function of the proton gradient is to displace Mg2+ from this site to permit cyclic repetition of the binding process. Essential for this scheme is the cyclic opening and closing of the proton channel. We have outlined our present approaches to test this hypothesis.

Structure and energy-linked activities in reconstituted bacteriorhodopsin-yeast ATPase proteoliposomes

Bacteriorhodopsin-F 1·F 0 (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F 1·F 0 is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F 1·F 0 by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [ 32P]ATP formation where pyridine is present during first stage illumination. F 1·F 0 can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.

Analysis of the protein subunit structure of the oligomycin sensitive ATPase proteolipid fraction by reverse phase high pressure liquid chromatography

The proteolipid fraction which is obtained from the bovine mitochondrial oligomycin sensitive ATPase complex by extraction with chloroform:methanol is resolvable into 7 components by preparative reverse phase high pressure liquid chromatography. Each of these 7 components is present together with 4 additionally resolved components in the proteolipid fraction which is obtained by extraction of submitochondrial particles with chloroform: methanol. Of the 7 components derived from the oligomycin sensitive ATPase, 4 have been identified with known protein subunits of the membrane sector of this complex, 2 are newly documented subunits of this complex, and 1 remains uncharacterized.

On the Oligomycin-Sensitivity and Subunit Composition of the ATPase Complex from Rhodospirillum rubrum

Abstract Two alternative procedures for isolation of the oligomycin-sensitive ATPase complex (E. C. 3.6.1.3) from R. rubrum chromatophores are compared with respect to ease, rapidity, and yield. The inhibitory effect of oligomycin on the membrane-bound Ca2+ -ATPase activity is increased during storage of the chromatophores, whereas the effect of oligomycin on the membrane-bound Mg2+ -ATPase activity does not change within a week. Oligomycin-sensitivity of the solubilized ATPase complex depends on the isolation procedure. The enzyme complex consists of at least nine different polypeptides with the apparent molecular weights of (1) 67,000, (2) 60,000, (3) 56,000, (4) 35,000, (5) 22,000, (6) 20,000, (7) 17,000, (8) 12,000, (9) 11,000. The polypeptides 2 - 4, 7, and 8 represent subunits of coupling factor 1.

Phosphate transport in yeast mitochondria: purification and characterization of a mitoribosomal synthesis dependent proteolipid showing a high affinity for phosphate.

It is possible to obtain from yeast mitochondria a proteolipid able to bind phosphate, by two different procedures. One of them, generally used for lipid extraction, leads to the preparation of a more active crude proteolipid. This crude proteolipid has been purified by various chromatographic procedures and the active fraction, in phosphate binding, is always associated with cardiolipin. Its molecular weight seems to be close to 10000. The phosphate binding shows ligand saturation behavior and is inhibited by arsenate and N-ethylmaleimide; succinate is noninhibitory. This protein seems to be dependent on the mitoribosomal synthesis since it is not present in mitochrondria of mutant "petite colonie" and its amount largely decreases in mitochondria from yeast grown in the presence of chloramphenicol. It is possible to extract a proteolipid from the oligomycin sensitive ATPase, showing the same activity and properties. The hypothesis that this proteolipid acts as a part of the Pi carrier and constitutes the oligomycin-sensitive ATPase complex is discussed.

Solubilization of an oligomycin-sensitive ATPase complex from Rhodospirillum rubrum chromatophores and its inhibition by various antibiotics

Solubilization of an oligomycin-sensitive ATPase complex from Rhodospirillum rubrum chromatophores and its inhibition by various antibiotics

Studies on the ATPase complex from beef-heart mitochondria. I. Isolation and characterization of an oligomycin-sensitive and an oligomycin-insensitive ATPase complex from beef-heart mitochondria

1. 1. A new method for the isolation of the oligomycin-sensitive ATPase from beef-heart mitochondria is described. 2. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg 2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 μmol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 μmol for the oligomycin-insensitive complex. 6. 6. The preparation of the oligomycin-sensitive complex contains at least 6–7 polypeptides in addition to those derived from F 1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. 7. Some of these polypeptides have been identified and isolated.

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.

Localization of genes for variant forms of mitochondrial proteins on mitochondrial DNA of Saccharomyces cerevisiae

Electrophoretic variant forms of mitochondrial translation products have been analyzed genetically and biochemically to determine their position on the yeast mitochondrial genome and to determine if they correspond to known products of mitochondrial protein synthesis. Three variant polypeptide species designated var1 ( M r = 40,000 to 42,000), var2 ( M r = 25,000), and var3 ( M r = 45,000) have been mapped by a combination of recombinational mapping by crosses to wild-typo testers and by deletion mapping procedures using petites of defined mitochondrial genotypes. In all cases, the variant polypeptide species have been mapped relative to each other and to known mitochondrial drug-resistance markers. We have not observed any recombinants between var1 and var2 and thus these polypeptide species maybe derived from a common transcriptional or translational precursor, var1 and var2 were found to map in the span between ery and oli1, at a site which represents a new locus for a mitochondrial structural gene. From immunological analysis and molecular weight considerations, we conclude that var1, whose function is unknown, is probably not a component of cytochrome oxidase, the oligomycin-sensitive ATPase complex or the bc 1 segment of the mitochondrial electron transport chain. var3 maps very close to and is probably allelic with the oli1 locus. We conclude that the var3 species, which shows metastable aggregation in the presence of sodium dodecyl sulfate, is a component of the oligomycin-sensitive ATPase complex, most likely subunit 9 of that complex.

Investigation of the structural arrangement of the protein subunits of mitochondrial ATPase

The structure of initochondrial ATPase from Saccharomyces cerevisiae was investigated to establish spatial relationships among the peptide components of this enzyme complex. Reagents which reversibly cross-linked adjacent polypeptides in complex structures were used to propose tentative assignments of neighboring polypeptides both in F 1 ATPase and in the oligomycin-sensitive ATPase complex. Two-dimensional gel electrophoresis of cross-linked and dissociated samples of the enzyme was used to analyze the sizes and subunit compositions of oligomers formed by cross-linking reactions. Two of the nine major peptide components of the ATPase complex were labeled with [ 3H]amino acids in the presence of cycloheximide. Analysis of association of these labeled components with other peptides of the complex was therefore facilitated by autoradiographic techniques. The cross-linking studies give rise to a suggested structural model for ATPase.

Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[(3)H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F(1)-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[(113)Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8-9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000-8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.

Studies of energy-linked reactions. Localization of the site of action of trialkyltin in yeast mitochondria.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.

Reconstitution of biological molecular generators of electric current. H+-ATPase.

1. Generation of a transmembrane electric potential difference by oligomycin-sensitive ATPase complex, incorporated into spherical or planar phospholipid membrane, has been demonstrated. To this end, penetrating anion probe and direct voltmeter measurement of electric potential across phospholipid membrane were used. It was found that ATP-induced electric response is sensitive to oligomycin and protonophorous uncouplers. 2. The effect of variations in the phospholipid component of proteoliposomes on the electric generation was studied. It was revealed that the usage of mitochondrial phospholipids and phosphatidylethanolamine allows the highest values of membrane potential to be obtained in the case of ATPase proteoliposomes. In the case of cytochrome oxidase and bacteriorhodopsin proteoliposomes, phosphatidylserine was also shown to be quite suitable. Phosphatidylcholine was absolutely ineffective in all cases. 3. In proteoliposomes, containing both ATPase and bacteriorhodopsin, ATP and light induced generation of the electric field of the same direction. 4. In ATPase + cytochrome oxidase proteoliposomes, ATP hydrolysis and ascorbate oxidation was found to support electric generation of the same direction if cytochrome c was inside vesicles. Oxidation via external cytochrome c resulted in formation of electric field of the direction, opposite to that induced by ATP hydrolysis. 5. The data obtained in experiments with proteoliposomes of different types are discussed. The conclusion is made that conversion of energy of different resources into electric form is a common feature of membraneous energy transducers, which is in agreement with the Mitchellian principle of cellular energetics.