Oligomycin Sensitivity-conferring Protein Research Articles

Membrane Topography and Near-neighbor Relationships of the Mitochondrial ATP Synthase Subunits e, f, and g

The well characterized subunits of the bovine ATP synthase complex are the alpha, beta, gamma, delta, and epsilon subunits of the catalytic sector, F1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivity-conferring protein), F6, and A6L, which are present in the membrane sector, F0, and the 45-A-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10. 2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e cross-links to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria.

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.

Fenazaquin Acaricide Specific Binding Sites in NADH: Ubiquinone Oxidoreductase and Apparently the ATP Synthase Stalk

Fenazaquin [4-(4-tert-butylphenethoxy)quinazoline] is one of several acaricides and insecticides, including rotenone and pyridaben, that are reported to act by inhibiting NADH:ubiquinone oxidoreductase (Complex I) (EC 1.6.99.3) in the range 1–10 nM. [3H]Fenazaquin is used here at 0.8 nMas a radioligand with electron transport particles (ETP) of bovine heart mitochondria to develop a new and rapid filtration assay with 42% specific binding (displaced by 2 μMrotenone). The potency of rotenoids, quinazolines, pyridaben, acetogenins, and several other disrupters of electron transport as inhibitors of NADH:ubiquinone oxidoreductase activity generally correlates with their effectiveness in displacing [3H]fenazaquin from its binding site in ETP (r2= 0.96,n= 18). This correlation extends to the fenazaquin analog with (trifluoromethyl)-diazirinyl replacing thetert-butyl substituent assayed at 6 nM,indicating that it also acts at a high-affinity site in Complex I. This diazirinyl analog of fenazaquin with tritium at theO-methylene position (4.7 Ci/mmol), on incubation at 435 nMwith ETP then irradiation at 350 nm, results in photoactivatable, irreversible specific binding which is fully protectable with rotenone. The site of labeling was examined by sequential isolation of the only band of specifically labeled protein(s) (which appeared at 22–24 kDa on SDS–PAGE),in situtryptic digestion of the protein(s) in the band, fractionation of the generated peptides by microbore HPLC, and amino acid sequence analysis. The single labeled HPLC peak of peptides in the tryptic digest was derived from two proteins, both components of ATP synthase, identified as subunit d and the oligomycin sensitivity conferral protein. An unusually low recovery of tryptophan at residue 12 of subunit d of specifically labeled ETP versus control ETP leads to the tentative proposal that labeling occurs at that site. Fenazaquin (and presumably rotenone as well) therefore has at least two specific binding sites in ETP, the anticipated high–affinity site in NADH:ubiquinone oxidoreductase and apparently a low-affinity site of unknown function, photoaffinity labeled in the stalk region of ATP synthase.

Oligomycin sensitivity conferring protein of mitochondrial ATP synthase: deletions in the N-terminal end cause defects in interactions with F1, while deletions in the C-terminal end cause defects in interactions with F0.

The structure/function relationships of oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase were studied by nested deletion mutagenesis, followed by analyses of the resultant OSCPs for their ability to restore partial reactions of ATP synthesis in OSCP-depleted F1-F0 complexes. Our results indicate that, from the N-terminus of OSCP, up to 13 amino acid residues could be deleted without any effect on OSCP coupling activity. However, deletion of 16 or more residues led to a slow decline in the ability of resultant mutant forms to restore ATP synthesis. Compared to the wild-type form of OSCP, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-Pi exchange activity. Detailed analyses of mutant ND-28 revealed that it was able to bind to the membrane segment (F0) of ATP synthase and restore oligomycin-sensitive ATPase activity in OSCP-depleted F1-F0 complexes. However, it did not bind to soluble segment F1, nor did it confer cold stability to either soluble F1 or reconstituted F1-F0 complex. On the other hand, studies on nested deletions on the C-terminal end indicate that three residues could be deleted without compromising the energy-coupling activity of OSCP. However, truncations of five or more residues caused an impairment in the ability of resultant mutant forms to restore ATP-Pi exchange activity in OSCP-depleted complexes. Mutant CD-10 (deletion of amino acids 181-190) was completely ineffective as a coupling factor. Detailed analyses of this mutant revealed that the subunit was able to bind to soluble F1 segment and confer cold stability to the enzyme but was neither able to associate with the membrane segment (F0) nor able to reconstitute high oligomycin sensitivity in depleted F1-F0 complexes. We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coupling factor to soluble F1 but not for coupling the energy of proton translocation to the synthesis of ATP; on the other hand, the carboxyl-terminal end of OSCP containing amino acids K181-M186 is important for F0-OSCP interactions as well as for the coupling of the energy of delta microH+ during the synthesis of ATP. These results suggest a model for OSCP in which the N-terminus is associated with the F1 segment and the C-terminus is associated with the F0 segment, while the central part of the polypeptide forms three or more helices constituting the stalk in the intact F1F0 enzyme.

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

Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP50 gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. ( Nature 359:380, 1992) YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP50 subunit is a key structural component of the stalk of the mitochondrial respiratory chain F 1F 0-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21).

The Functional Expression of a Rat cDNA Encoding OSCP in the Yeast Saccharomyces cerevisiae

The oligomycin sensitivity conferring protein (OSCP) of Saccharomyces cerevisiae is a component of the stalk F A sector of the mitochondrial F 1F O-ATP synthase complex (mtATPase). The subunits comprising this sector have been implicated in the coupling of proton flux through the membrane F O sector to ATP synthesis on the catalytic F 1 sector. We tested whether the yeast subunit encoded by the ATP5 gene could be functionally replaced by a mammalian homologue, rat OSCP. The yeast and rat OSCP proteins have 58% homology in terms of conserved amino acids. A vector-borne rat cDNA encoding the OSCP precursor was introduced into a yeast strain lacking endogenous OSCP as a result of its disrupted ATP5 gene. The resultant yeast cells showed essentially a wildtype growth rate at 18, 28 and 35°C, on nonfermentable substrates. Expression of mammalian F A subunit homologues in yeast should provide a productive new approach to generating information on the structure and function of mtATPase.

The ATP synthase (F 1F o) of Streptomyces lividans: sequencing of the atp operon and phylogenetic considerations with subunit β

The DNA encoding the subunits of the ATP synthase (F 1F o) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit γ of the F 1 complex. The complete nucleotide sequence of the operon was determined. The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function. The gene order found is identical to that in other non-photosynthetic eubacteria. The determination of the N-terminal amino acid (aa) sequences of the F 1 subunits α, β, γ, δ and ε allowed us to identify the translational start points and to define the primary structures of the proteins. The aa sequence deduced for subunit δ revealed an N-terminal extension of about 90 aa, which is not present in any δ subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far. The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved β subunit of different ATP synthases including that of S. lividans. The calculations revealed that S. lividans β does not form a phylogenetic group together with the Gram + taxa of low G+C contents, but is more closely related to the β subunit of Rhodobacteria.

ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

ATP Synthase from Bovine Heart Mitochondria In Vitro Assembly of a Stalk Complex in the Presence of F1-ATPase and in its Absence

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli . They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b′, bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment ′ (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b′, bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1·OSCP·b′, F1-OSCP-bI or F1·OSCP·bC were formed, but b′, bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1·OSCP·b′·F6 and F1·OSCP·bI·F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1·OSCP·b′·F6·d, F1·OSCP·b′·F6·′ and F1·OSCP·b·F6·d were isolated. In a further series of reconstitution experiments, the binary complexes b′·OSCP and b′·d, the ternary complex b′·′·F6, and the quaternary complex OSCP·b′·F6·d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1·stalk complex, F1·OSCP·b′·d·F6, each contained one copy per complex of subunits b (or b′), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1 : 1 : 1. The ratio of b to d in purified F0 was 1 : 1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

ATP Synthase from Bovine Heart Mitochondria: In Vitro Assembly of a Stalk Complex in the Presence of F1-ATPase and in its Absence

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli . They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b′, bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment ′ (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b′, bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1·OSCP·b′, F1-OSCP-bI or F1·OSCP·bC were formed, but b′, bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1·OSCP·b′·F6 and F1·OSCP·bI·F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1·OSCP·b′·F6·d, F1·OSCP·b′·F6·′ and F1·OSCP·b·F6·d were isolated. In a further series of reconstitution experiments, the binary complexes b′·OSCP and b′·d, the ternary complex b′·′·F6, and the quaternary complex OSCP·b′·F6·d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1·stalk complex, F1·OSCP·b′·d·F6, each contained one copy per complex of subunits b (or b′), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1 : 1 : 1. The ratio of b to d in purified F0 was 1 : 1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

Low Levels of ATP Synthase and Cytochrome c Oxidase Subunit Peptide from Hearts of Copper-Deficient Rats Are Not Altered by the Administration of Dimethyl Sulfoxide

This study determined if reported decreases in the delta subunit of ATP synthase and nuclear-encoded cytochrome c oxidase subunits in hearts of copper-deficient rats were secondary to the heart disease pathology or due to lack of the trace element. Male weanling Long-Evans rats were randomly divided into six groups: rats fed a copper-adequate or copper-deficient diet (with free access) with or without 5% dimethyl sulfoxide (DMSO) in the drinking water and rats pair-fed the copper-adequate or copper-deficient diet without DMSO treatment. After 4 wk, rats in the groups fed the copper-deficient diet had lower liver superoxide dismutase and heart cytochrome c oxidase activities compared with groups fed the copper-adequate diet. Administration of DMSO, an antioxidant, and energy restriction (pair-feeding) partially blocked cardiac hypertrophy in rats fed the copper-deficient diet. Greater mitochondrial volume density and mitochondrial:myofibrillar ratio and disrupted myofibrils and basal laminae were observed in the hearts from rats fed the copper-deficient diet and not treated with DMSO compared with hearts from groups fed the copper-adequate diet. The DMSO-treated rats fed the copper-deficient diet had hearts with intact structure but enlarged mitochondria compared with other groups fed the copper-deficient diet. The delta subunit of ATP synthase and the nuclear-encoded cytochrome c oxidase subunits IV and V were depressed in rats fed a copper-deficient diet regardless of antioxidant treatment and pair-feeding. These data suggest that the effects of copper deficiency upon ATP synthase and cytochrome c oxidase proteins are not due to the cardiac pathology.

Cross-reconstitution studies with polypeptides of Escherichia coli and bovine heart mitochondrial F0F1 ATP synthase.

To characterize the role of supernumerary subunits of the mammalian F0F1 ATP synthase, cross-reconstitution of mitochondrial and bacterial F0F1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian F0, in particular F0I-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N,N'-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N,N'-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial F0I-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. F0I-PVP and OSCP added to UPEc, promoted inhibition by N,N'-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli F0. The presence of F0I-PVP and OSCP also promoted inhibition by N,N'-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.

ATP synthesis and hydrolysis of the ATP-synthase from Micrococcus luteus regulated by an inhibitor subunit and membrane energization

After incubation for 70 min in Tris-HCl (pH 8.0), the rate of ATP hydrolysis of free and reconstituted ATP-synthase from Micrococcus luteus multiplied about three times. The apparent increase in activity is due to the reversible dissociation of the δ-subunit. Results of experiments on the temperature dependence of the ATP hydrolysis rate of substrate saturated ATP-synthase exhibited a discontinuity in the Arrhenius plot at 32 ± 0.5°C for the δ-subunit associated enzyme. Below 32 ± 0.5°C the activation energy, E a, was 231.5 ± 5 kJ mol −1, while above this temperature-level it decreased to 76.4 ± 3 kJ mol −1. ATP synthesis and hydrolysis of the ATP-synthase, co-reconstituted with monomeric bacteriorhodopsin ( Halobacterium halobium), showed a lag of 50 s upon the illumination with green light (505–575 nm). This retardation and the activity depended on the ATP-synthase concentration, being typical of the dissociation of an inhibitor protein. The N-terminal protein sequences of the δ-and ϵ-subunit of the ATP-synthase were identified by automated Edman degradation. Alignment of the amino acid sequence and secondary structure calculations for the δ-subunit did not reveal homology to other known ATP-synthase δ-subunits, but significant equivalence to the ϵ-subunit of E. coli. Sequence analysis of the ϵ-subunit from M. luteus showed homology to equivalent regions in δ-subunits and Oligomycin Sensitivity Conferring Protein (OSCP) of other organisms.

Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media

Two independent cold-sensitive pet mutants in the gene (ATP5) coding for the oligomycin sensitivity conferring protein (OSCP) have been isolated in the yeast Saccharomyces cerevisiae. The mutations in both strains alter the initiating methionine codon in the ATP5 gene: ATG to ATA (Ile) and AAG (Lys). Western blot analysis of total yeast protein after the cells were grown at 18°C, 30°C, and 37°C, indicates that the level of OSCP decreased 80% relative to the wild type strain. In addition, the level of the oligomycin-sensitive ATPase decreased 85% relative to the wild type strain, after growth at 30°C. These findings indicate that for S. cerevisiae, the level of oxidative phosphorylation can decrease 85% without showing a large growth defect on media containing glycerol at 30°C, but not at 18°C.

A Unified Perspective on Copper Deficiency and Cardiomyopathy

Dietary copper restriction in rats results in cardiomyopathy. In rats fed copper-restricted diets from weaning for 5 to 8 weeks, a concentric hypertrophy is apparent, whereas postweaning copper restriction does produce cardiomyopathy without apparent hypertrophy. Both sets of circumstances appear to affect the integrity of the basal laminae of cardiac myocytes and capillaries. In rats fed copper-restricted diets from weaning, decreases in cytochrome c oxidase are related not only to copper's role as a coenzyme, but also to a marked decrease in the nuclear encoded subunits of the enzyme complex. Decreased levels of the delta-subunit of ATP synthase have been observed. However, such aberrations in mitochondrial enzymes, as well as morphologic alterations, apparently do not affect cardiac levels of ATP. This review suggests mechanisms of cardiac adaptation and initiation factors leading to cardiac hypertrophy. We present a hypothetical working model explaining the events leading to cardiac failure in the copper-deficient rat heart based on the present body of knowledge, and compare the pathology with other models of cardiomyopathies.

Molecular cloning and sequence of cDNAs for the import precursors of oligomycin sensitivity conferring protein, ATPase inhibitor protein, and subunit c of H +-ATP synthase in rat mitochondria

Four cDNAs for the import precursors of oligomycin sensitivity conferring protein (OSCP), ATPase inhibitor protein (IF 1) and subunit cs (encoded by P1 and P2 genes) of rat mitochondrial H +-ATP synthase have been cloned from a rat cDNA library. The import precursors and the mature polypeptides of rat OSCP, IF 1, subunit c (P1) and subunit c (P2) consisted of 23 190 , 25 82 , 61 75 and 66 75 amino acids, respectively.

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.

Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.

Oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase. The carboxyl-terminal region of OSCP is essential for the reconstitution of oligomycin-sensitive H(+)-ATPase.

Studies to establish the structure/function relationships of oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase were carried out using genetic engineering and biochemical approaches. A full-length cDNA clone encoding OSCP was isolated from a bovine heart cDNA library, and the mature form of OSCP was expressed in Escherichia coli using plasmid expression vector pKP1500. Recombinant OSCP was found to accumulate in the cytoplasmic inclusion bodies, by virtue of which the recombinant protein could be purified to greater than 85% purity by simple low speed centrifugation of cell lysates. Recombinant OSCP was found to be indistinguishable from OSCP isolated from mitochondria with respect to (i) apparent molecular mass on sodium dodecyl sulfate gel electrophoresis, (ii) immunological reactivity to anti-OSCP serum, (iii) biological activity in restoring oligomycin-sensitive ATPase and Pi-ATP exchange activities to OSCP-depleted ATP synthase complexes, and (iv) insensitivity of the biological activity to sulfhydryl-directed alkylating reagents. The amino-terminal sequence of the recombinant protein revealed that the initiating methionine was not removed by E. coli, although that apparently did not affect protein folding or its biological activity. Data on nested deletion mutations starting from the carboxyl terminus in OSCP demonstrated that, in each instance, the mutant form was expressed and the protein product was sequestered in cytoplasmic inclusion bodies, similar to the wild-type form. However, none of the variants, including the one in which only the last 10 residues were deleted, was able to restore cold-stable oligomycin-sensitive ATPase or Pi-ATP exchange activity in OSCP-depleted complexes. Taken together, these data suggest that amino acid residues 181-190 (or some of the residues in this region) in the OSCP sequence may be important for OSCP-F1 interactions.

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.