Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.     

The biogenesis of a cytochrome b complex in yeast mitochondria: Sites of translation of the protein components

A spectrally pure cytochrome b complex has been isolated from yeast mitochondria and shown to contain seven nonidentical subunits with the following molecular weights: I, 42,000; II, 33,000; III, 27,500; IV, 23,000; V, 15,500; VI, 13,000; and VII, 10,500. In order to determine the intracellular sites of translation of these polypeptides, yeast cells were labeled with [³H]leucine in the presence of specific inhibitors of mitochondrial or cytoplasmic translation. The labeling of subunits I and III was found to be insensitive to cycloheximide but was inhibited by chloramphenicol. Alternatively, subunits IV–VII were labeled in the presence of chloramphenicol but not in the presence of cycloheximide. Since subunit II was not significantly labeled in the presence of either inhibitor, the technique of labeling in vivo with [³H]formate was used to establish its site of biogenesis. Formate is incorporated by mitochondrial, but not cytoplasmic, ribosomes as N-formylmethionine at initiation and is therefore a marker for the products of mitochondrial translation. Subunits I–III were labeled under these conditions whereas the four smallest subunits were not. Taken together, the findings clearly establish that the three largest subunits of the cytochrome b complex are translated on mitochondrial ribosomes and that the four smallest are formed in the cytoplasm. The results also underscore the advantages of using [³H]formate to identify the products of mitochondrial translation.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.     

Synthesis and processing of glycoprotein D of herpes simplex virus types 1 and 2 in an in vitro system.

We carried out studies of in vitro translation and processing of glycoprotein D (gD) of herpes simplex virus types 1 and 2 by using mRNA from cells infected for 6 h and a reticulocyte lysate translation system. Polypeptides of 49,000 daltons were immunoprecipitated with anti-gD-1 sera. Each in vitro-synthesized molecule had the same methionine tryptic peptide profile as the respective in vivo precursors, pgD-1 and pgD-2. In addition, the polypeptides synthesized in vitro were larger than the corresponding molecules synthesized in the presence of tunicamycin. This suggested that each of the gD polypeptides synthesized in vitro contained a transient N-terminal signal sequence. When the translation mixture was supplemented with pancreatic microsomes, each of the gD polypeptides was converted cotranslationally to a larger-molecular-weight form. Processing involved addition of three N-asparagine-linked oligosaccharides and removal of the signal peptide. When trypsin was added after in vitro processing, a polypeptide which was 3,000 daltons smaller than the in vitro-modified form of gD was immunoprecipitated. Experiments with endo-beta-N-acetylglucosaminidase H showed that this polypeptide still contained the three N-asparagine-linked oligosaccharides. Two monoclonal antibodies, 57S (group V) and 17O (group VII), were used to further orient gD in microsomes. The group V determinant was located in the trypsin-sensitive 3,000-dalton fragment, and the group VII determinant was located in the portion of gD which was protected from trypsin. We concluded that gD is oriented with the three glycosylation sites inside the vesicles and that 3,000 daltons containing the group V determinant are located outside. Immunofluorescence studies indicated that the group V determinant of gD is inside the plasma membrane of herpes simplex virus-infected cells and that the group VII determinant is outside. This cellular orientation is consistent with predictions based on the in vitro experiments.     

Isolation of the cytochrome-bc1 complex from rat-liver mitochondria

The cytochrome bc1 complex has been isolated from rat-liver mitochondria by two different procedures. The enzyme isolated by either procedure exhibits a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg protein respectively. Both preparations contain only seven polypeptides on sodium dodecylsulfate gel electrophoresis, with the following apparent molecular weights: I, 50000; II, 46000; III, 33000; IV, 25000; V, 12500; VI, 10000; VII, 5600. The polypeptide composition is identical to that of the beef-heart enzyme isolated by cholate/ammonium sulfate fractionation. Furthermore, with the exception of subunit II (core protein 2), the apparent molecular weights of the subunits are identical in the rat-liver and beef-heart enzymes.     

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.     

The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.     

A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the resolution of cytochrome oxidase

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I–IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V–VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of hemea ₃. It is suggested that the fraction of subunits I–IV, VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.     

Heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits in Saccharomyces cerevisiae.

The presence of cytochrome c oxidase subunits and the association of these subunits with each other was studied in a heme-deficient Saccharomyces cerevisiae mutant. This mutant had been isolated by Gollub et al. (1977) J. Biol. Chem. 252, 2846-2854) and had been shown lack delta-aminolevulinic acid synthetase. When grown in the absence of heme or heme precursors, the mutant is respiration-deficient, devoid of cytochrome absorption bands and auxotrophic for all those components whose biosynthesis is dependent on hemoproteins; when grown in the presence of heme or heme precursors, the mutant is phenotypically wild type. Upon growth of the mutant in the absence of heme synthesis, the mitochondria still contained two of the three mitochondrially made cytochrome c oxidase subunits (i.e. II and III) and at least one of the cytoplasmically made cytochrome c subunits (VI). The other subunits were either barely detectable (I, IV) or undetectable (V, VII). The residual subunits were apparently not assembled with each other since an antiserum directed mainly against Subunit VI failed to co-precipitate Subunits II and III which were still present. In contrast, growth of the mutant in the presence of delta-aminolevulinic acid led to the accumulation of active, fully assembled cytochrome c oxidase in the mitochondria. Heme a (or one of its precursors) thus controls the assembly of cytochrome c oxidase from its individual subunits.     

Role of exogenous hemin in the synthesis of hemoproteins and nonheme proteins during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin to glucose-repressed cells of Saccharomyces cerevisiae stimulates the incorporation of amino acid into cytoplasmic proteins twofold. There was no significant change in the synthesis of total cytoplasmic RNA whereas a 40% increase in the synthesis of poly(A)-containing RNA was observed upon hemin treatment. Cell-free translation of cytoplasmic mRNAs and immunoprecipitation analysis of the translated products with antibodies against subunit V of cytochrome oxidase and the alpha and beta subunits of F1-ATPase reveals that there is an eightfold enrichment of the mRNA for subunit V of cytochrome oxidase upon hemin treatment. The effect of hemin on the alpha and beta subunits of F1-ATPase is only marginal, suggesting a differential role for heme in the synthesis of hemoproteins and nonheme proteins during glucose repression.     

Interactions among the three adenovirus core proteins.

Interactions among the three adenovirus core polypeptides V, VII, and mu were examined, using the reversible chemical cross-linker dithiobis(succinimidyl propionate) and two-dimensional polyacrylamide gel electrophoresis. Cross-linked species obtained from gradient-purified adenovirus type 2 cores were well represented among the cross-linked products of pentonless virions and crude core preparations. The more efficiently formed cross-linked core species were also identified with the arginine-specific cross-linker, p-azidophenyl glyoxal. In addition to dimers of polypeptides V and VII, efficient cross-linking of V to VII, V to mu, and VII to V to mu was detected in adenovirus cores. Notably absent were cross-linked species corresponding to higher multimers of polypeptide VII. A major core-capsid interaction appeared to be via the association of polypeptide V with a dimer of polypeptide VI.     

Regulation of the nuclear-coded peptides of yeast cytochrome c oxidase

We have analyzed the catabolite regulation of cytochrome oxidase by assaying changes in the synthesis of precursors of the nuclear-coded peptides (IV--VII) of cytochrome c oxidase in an in vitro reticulocyte cell-free system programmed with RNA isolated from cells grown in either glucose or raffinose. As a first step, we have characterized antibodies which bind to the precursors of subunits V and VI. Initial translation products for subunits IV and VII have also been tentatively identified by utilizing these antibodies. The messenger RNAs coding for the precursors of the nuclear-coded subunits fall in the expected size range of 8--15 S. Catabolite repression of the nuclear-coded oxidase peptides appears to be regulated by the abundance of their messenger RNAs. Translation of messenger RNA isolated from yeast cells grown on glucose indicates a coordinate and uniform increase in precursor synthesis during glucose derepression. In contrast, when RNA isolated from raffinose (derepressed) grown cells is used to direct cell-free translation, precursor abundance is high throughout growth, although the synthesis of some of the species changes in a complex pattern of ratio and abundance. These data indicate that the abundance of the messengers for the nuclear-coded precursors is regulated in a fashion dependent on the physiologic state of the cell.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity.

In order to study the role of the individual subunits of yeast cytochrome c oxidase, rabbit antisera were prepared against Subunit II (a mitochondrially made polypeptide) and Subunit VI (a cytoplasmically made polypeptide). Antisera were also obtained against a mixture of the two mitochondrially made subunits (I PLUS II) and against mixtures of the following cytoplasmically made subunits: (IV PLUS VI); (V PLUS VII); and (IV PLUS V PLUS VI PLUS VII). Neither anti-II serum nor anti-VI serum cross-reacted with any of the other six subunits of cytochrome c oxidase as judged by a sensitive ring test or by double diffusion in agarose gels. Anti-II serum inhibited the oxidation of ferrocytochrome c by purified yeast cytochrome c oxidase or by freshly isolated as well as sonically fragmented yeast mitochondria. Anti-(V, VII) serum and anti-(IV, V, VI, VII) serum were also strongly inhibitory. Anti-VI serum and anti-(IV, VI) serum inhibited only weakly. If purified cytochrome c oxidase was inhibited with a saturating amount of anti-VI serum, anti-II serum elicited a further increment of inhibition, as would be expected if the inhibitory effects of these two antisera involved different antigenic sites on the holoenzyme. Each of the antisera precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. However, anti-VI and, particularly, anti-II were much less effective precipitants than antisera against Subunits IV to VII or antisera against the holoenzyme. These data suggest that the oxidation of ferrocytochrome c by cytochrome c oxidase required both mitochondrially as well as cytoplasmically made subunits.     

In Vitro Synthesis of Adenovirus Core Proteins

mRNA extracted from polysomes of KB cells at late stages of productive infection with adenovirus type 2 was translated in a cell-free system derived from Krebs II ascites cells. Two of the polypeptides obtained in this reaction corresponded to the adenovirus core protein V and the precursor to core protein VII. The following criteria were used to establish identity between the in vitro products and the virion proteins: comigration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis, cochromatography in sodium dodecyl sulfate-hydroxyapatite, specific immunoprecipitation of the precursor to core protein VII, and tryptic peptide analysis.     

In vitro translation with adenovirus polyribosomes.

Polyribosomes isolated from adenovirus type 2 (Ad2)-infected HeLa cells late in productive infection can be used for translation in cell-free systems. At least eight viral polypeptides are synthesized, including the precursors to virion polypeptides VI and VII. Separation of polyribosomes by zonal rate centrifugation followed by translation in a cell-free system reveals a correlation between the sizes of the polyribosomes and the polypeptides synthesized. The cell-free extracts incorporate amino acid linearly for only 10 min and show little or no capacity to reinitiate protein synthesis. The elongation efficiency measured as the number of amino acids incorporated per ribosome in 20 min is low, ranging from 10 to 100. The maximum chain elongation rate is estimated to be 10 to 20 amino acids per min. The limited elongation has been used to assess the relative concentration of mRNA's engaged in translation.     

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.