Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.     

COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae

We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies.     

Modular assembly of yeast cytochrome oxidase

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high–molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.     

The assembly pathway of complex I in Arabidopsis thaliana

All present‐day mitochondria originate from a single endosymbiotic event that gave rise to the last eukaryotic common ancestor more than a billion years ago. However, to date, many aspects of mitochondrial evolution have remained unresolved. Comparative genomics and proteomics have revealed a complex evolutionary origin for many mitochondrial components. To understand the evolution of the respiratory chain, we have examined both the components and the mechanisms of the assembly pathway of complex I. Complex I represents the first enzyme in the respiratory chain, and complex I deficiencies have dramatic consequences in both animals and plants. The complex is located in the mitochondrial inner membrane and possesses two arms: one embedded in the inner membrane and one protruding in the matrix. Here, we describe the assembly pathway of complex I in the model plant Arabidopsis thaliana. Using a proteomics approach called complexome profiling, we have resolved the different steps in the assembly process in plants. We propose a model for the stepwise assembly of complex I, including every subunit. We then compare this pathway with the corresponding pathway in humans and find that complex I assembly in plants follows a different, and likely ancestral, pathway compared with the one in humans. We show that the main evolutionary changes in complex I structure and assembly in humans occurred at the level of the membrane arm, whereas the matrix arm remained rather conserved.     

Cis-regulation of inter-allelic exchanges in mutation at human minisatellite MS205 in yeast.

Tandemly repeated DNA is a major component of the human genome, and includes loci contributing to human disease. Minisatellites include the most variable human loci described to date, and the mechanisms by which this variation is generated in humans have been studied in detail. Integration of human minisatellites into yeast not only provides a model for further dissecting the molecular basis of length change mutation at these loci, but also more generally allows the study of complex recombinational events in yeast. We have used human minisatellite MS205 integrated into yeast to study the structural details of length change mutations. Apart from showing that mutation at this locus in yeast has features similar to those observed at some minisatellites in humans, including meiosis-specificity, and polarity, in which exchange events are localised to one extremity of the array, we here, for the first time, directly demonstrate that a flanking element in yeast regulates the mutation process. The results therefore support the hypothesis that flanking initiators are involved in minisatellite mutation in humans. Furthermore, mutant alleles showed more complex rearrangements in one orientation than the other. The data also suggest that the mutational pathway for deletions might be different from the pathway generating inter-allelic exchanges and duplications.     

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.     

Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.     

Cytochrome c oxidase deficiency: Patients and animal models

Cytochrome c oxidase (COX) deficiencies are one of the most common defects of the respiratory chain found in mitochondrial diseases. COX is a multimeric inner mitochondrial membrane enzyme formed by subunits encoded by both the nuclear and the mitochondrial genome. COX biosynthesis requires numerous assembly factors that do not form part of the final complex but participate in prosthetic group synthesis and metal delivery in addition to membrane insertion and maturation of COX subunits. Human diseases associated with COX deficiency including encephalomyopathies, Leigh syndrome, hypertrophic cardiomyopathies, and fatal lactic acidosis are caused by mutations in COX subunits or assembly factors. In the last decade, numerous animal models have been created to understand the pathophysiology of COX deficiencies and the function of assembly factors. These animal models, ranging from invertebrates to mammals, in most cases mimic the pathological features of the human diseases.     

Cytochrome c oxidase biogenesis: new levels of regulation

Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are posttranslationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed.     

Yeast models of human mitochondrial diseases

The yeast Saccharomyces cerevisiae is an excellent model for gaining insights into the molecular basis of human mitochondrial disorders, particularly those resulting from impaired mitochondrial metabolism. Yeast is a very well characterized system and most of our current knowledge about mitochondrial biogenesis in humans derives from yeast genetics and biochemistry. Systematic yeast genome-wide approaches have allowed for the identification of human disease genes. In addition, the functional characterization of a large number of yeast gene products resident in mitochondria has been instrumental for the later identification and characterization of their human orthologs. Here I will review the molecular and biochemical characterization of several mitochondrial diseases that have been ascribed to mutations in genes that were first found in yeast to be necessary for the assembly of the mitochondrial respiratory chain. The usefulness of yeast as a model system for human mitochondrial disorders is evaluated.     

Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast

Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo-COX in the SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D-PAGE.     

New perspectives on the assembly process of mitochondrial respiratory chain complex cytochrome c oxidase

The assembly of cytochrome c oxidase (COX) is a complicated process and requires a number of assembly factors to put all the necessary subunits in the correct position. Defects in COX assembly lead in particular to serious neuromuscular disorders. We demonstrated that COX-deficient patients can be associated with different assembly patterns. To obtain more insight in the biogenesis of COX in a living cell, we used yeast as a model organism to design a way to pulse label holo-COX with green fluorescent protein (GFP). Using blue native electrophoresis, we showed that the GFP-tagged subunit is incorporated into fully assembled COX and this GFP tagged complex still has enzymatic activity. This allows us to correlate the GFP fluorescence signal detected in vivo by microscopy with the synthesis, turnover and assembly of COX.     

Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly regulated. In addition to the structural subunits, a large number of accessory factors are required to build the holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly defects, reviewed here, have become a profitable strategy to gain insight into their functions and the pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency.     

Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process

Cytochrome c-oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in the regulation of aerobic production of energy. Biogenesis of eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a protective shield surrounding the core. COX biogenesis requires the assistance of >20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclear-encoded subunits, insertion of the structural subunits into the mitochondrial inner membrane, addition of prosthetic groups, assembly of the holoenzyme, further maturation to form a dimer, and additional assembly into supercomplexes are all tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such regulation ensures the building of a highly efficient machine able to catalyze the safe transfer of electrons from cytochrome c to molecular oxygen and ultimately facilitate the aerobic production of ATP. In this review, we will focus on describing and analyzing the present knowledge about the different regulatory checkpoints in COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable yeast model, but also from bacterial and animal systems, by means of large-scale genetic, molecular biology, and physiological approaches and by integrating information concerning individual elements into a cellular system network.     

Early developmental pathology due to cytochrome c oxidase deficiency is revealed by a new zebrafish model

Deficiency of cytochrome c oxidase (COX) is associated with significant pathology in humans. However, the consequences for organogenesis and early development are not well understood. We have investigated these issues using a zebrafish model. COX deficiency was induced using morpholinos to reduce expression of CoxVa, a structural subunit, and Surf1, an assembly factor, both of which impaired COX assembly. Reduction of COX activity to 50% resulted in developmental defects in endodermal tissue, cardiac function, and swimming behavior. Cellular investigations revealed different underlying mechanisms. Apoptosis was dramatically increased in the hindbrain and neural tube, and secondary motor neurons were absent or abnormal, explaining the motility defect. In contrast, the heart lacked apoptotic cells but showed increasingly poor performance over time, consistent with energy deficiency. The zebrafish model has revealed tissue-specific responses to COX deficiency and holds promise for discovery of new therapies to treat mitochondrial diseases in humans.     

Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment

The COX17 gene of Saccharomyces cerevisiae codes for a cytoplasmic protein essential for the expression of functional cytochrome oxidase. This protein has been implicated in targeting copper to mitochondria. To determine if Cox17p is present in mammalian cells, a yeast strain carrying a null mutation in COX17 was transformed with a human cDNA expression library. All the respiratory competent clones obtained from the transformations carried a common cDNA sequence with a reading frame predicting a product homologous to yeast Cox17p. The cloning of a mammalian COX17 homolog suggests that the encoded product is likely to function in copper recruitment in eucaryotic cells in general. Its presence in humans provides a possible target for genetically inherited deficiencies in cytochrome oxidase. Received: 22 August 1996 / Revised: 31 October 1996     

Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.     

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.     

A mutation in C2orf64 causes impaired cytochrome c oxidase assembly and mitochondrial cardiomyopathy

The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy.     

Use of yeast as a system to study amyloid toxicity

The formation of amyloid-like fibrils is a hallmark of several neurodegenerative diseases. How the assembly of amyloid-like fibrils contributes to cell death is a major unresolved question in the field. The budding yeast Saccharomyces cerevisiae is a powerful model organism to study basic mechanisms for how cellular pathways regulate amyloid assembly and proteotoxicity. For example, studies of the amyloidogenic yeast prion [RNQ(+)] have revealed novel roles by which molecular chaperones protect cells from the accumulation of cytotoxic protein species. In budding yeast there are a variety of cellular assays that can be employed to analyze the assembly of amyloid-like aggregates and mechanistically dissect how cellular pathways influence proteotoxicity. In this review, we describe several assays that are routinely used to investigate aggregation and toxicity of the [RNQ(+)] prion in yeast.