Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼ 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9–10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.     

Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb₃-type cytochrome c oxidases (cbb₃-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa₃-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb₃-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b₃-Cu_B center, have to be coordinated precisely both temporally and spatially to yield a functional cbb₃-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb₃-Cox, and provide a highly tentative model for cbb₃-Cox assembly and formation of its heme b₃-Cu_B binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.     

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.     

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)     

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8_m reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.     

The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.     

Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria

Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

Dual Functions of Mss51 Couple Synthesis of Cox1 to Assembly of Cytochrome c Oxidase in Saccharomyces cerevisiae Mitochondria

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5′-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process.     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

Biogenesis of cbb(3)-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb(3)-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b(3)-Cu(B) center, have to be coordinated precisely both temporally and spatially to yield a functional cbb(3)-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb(3)-Cox, and provide a highly tentative model for cbb(3)-Cox assembly and formation of its heme b(3)-Cu(B) binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.