Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex

Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.     

Biosynthesis of cytochrome c oxidase by isolated rat liver mitochondria.

When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo.     

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

Site of synthesis of the mitochondrial cytochromes in hepatocyte cultures

The biosynthesis of mammalian mitochondrial cytochromes was explored in primary hepatocyte cultures. When these were pulsed with [35S]methionine in the presence of cycloheximide, eight discrete mitochondrial polypeptides were detected by fluorography after their resolution under denaturing conditions by polyacrylamide gel electrophoresis. Since the pulse labeling of the polypeptides was sensitive to chloramphenicol, an inhibitor of mitochondrial translation, they must be translated on mitochondrial ribosomes. Three were identified as the largest subunits of cytochrome oxidase by their immunoprecipitation with antibody directed against purified rat liver cytochrome oxidase. Another (Mr = 28,000) was identified as one of eight subunits of purified rat liver cytochrome b-c1 complex by its immunoprecipitation with antibody directed against bovine heart b-c1 complex. Since cytochrome b apoprotein is the only product of the mitochondrial genome in the yeast cytochrome b-c1 complex (Krieke, J., Bechmann, H., van Hemert, F. J., Schweyan, R. J., Boer, P. H., Kaudewitz, F., and Groot, G. S. P. (1979) Eur. J. Bio-chem. 101, 607-617), the results strongly suggest that the Mr = 28,000 subunit of liver b-c1 complex is cytochrome b apoprotein. Thus the contribution of the mitochondrial translation system to the cytochrome complexes in liver is identical to that of yeast and Neurospora, and there appears to be no deletion or transfer to the nuclear genome of structural genes for mitochondrially synthesized cytochromes during eukaryotic evolution.     

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[³⁵S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.     

Identification of cytochrome c oxidase subunits in nuclear yeast mutants lacking the functional enzyme.

Yeast mutants specifically lacking cytochrome c oxidase activity were screened for cytochrome c oxidase subunits by one- and two-dimensional electrophoresis, electrophoresis in exponential gradient gels, and immunoprecipitation with antisera against one or more of the cytoplasmically made subunits of the enzyme. Two cytochrome c oxidase-less nuclear mutants previously described from this laboratory each lack one or more mitochondrially synthesized cytochrome c oxidase subunits while possessing all four cytoplasmically synthesized subunits of that enzyme. The subunits remaining in these mutants were not assembled with each other; the cytoplasmically made subunits IV and VI could be released from the mitochondria by sonic oscillation, in contrast to the situation in wild type cells. No electrophoretically detectable alterations were found in any of the cytochrome c oxidase subunits present in the mutants. Nuclear mutations may thus cause both a loss as well as a defective assembly of mitochondrially made cytochrome c oxidase subunits.     

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.     

Synthesis of cytochrome oxidase in isolated rat hepatocytes

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharose-protein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase.     

On the molecular weight of mitochondrially synthesized subunits of rat liver cytochrome oxidase.

The subunit composition of cytochrome c oxidase from rat liver mitochondria was studied by dodecylsulfate polyacrylamide gel electrophoresis. The apparent molecular weight of the seven subunits are in reasonable agreement with published data on cytochrome c oxidase subunits from other sources. Two additional subunits were found if the electrophoresis was performed with 8m urea, due to splitting of the smallest subunit. Performic acid oxidation of the isolated subunits I and II increased the apparent molecular weights from 38000 to 48000 and from 24500 to 29000, respectively, accompained by a normalization of the anomalous behaviour of subunit I in the Ferguson plot. It is suggested that performic acid, by splitting extremely inaccessible disulfide bridges, mediates full complexing of the subunits by dodecylsulfate, thus permitting the determination of the real molecular weights by dodecylsulfate polyacrylamide gel electrophoresis.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.     

Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures

The degradation rates of inner mitochondrial membrane proteins were examined in serum-deprived hepatoma cultures. In those nonproliferating cells, the degradation of composite mitochondrial proteins was a first order process with a half-life of 34 h. The half-lives of specific inner mitochondrial membrane polypeptides were determined by examining the 3H/35S of isolated polypeptides from cells given [3H]methionine and [35S]methionine pulses, respectively, before and after a 2-day chase period. The 33 most abundant polypeptides resolved on a bidirectional polyacrylamide gel system showed half-lives ranging from 20 to 100+ h. The 15 polypeptides translated on mitochondrial ribosomes in the presence of inhibitory concentrations of cycloheximide also displayed heterogeneous rates of degradation (t1/2 = 35-100+ h). None of the isolated adenosine triphosphatase (coupling factor F1) or immunoprecipitated cytochrome c oxidase subunits were significantly turned over during the case period. Five of eight cytochrome b-c1 complex subunits, however, were turned over significantly more rapidly (t1/2 = 39-42 h) than the other three (t1/2 = 94+ h). The results demonstrate heterogeneous degradation rates for inner membrane polypeptides, extending in some cases to those within the same respiratory complex.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Proteolytic precursor processing in the biosynthesis of mitochondria

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.     

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c₁ subunits and by inference from comigration.