Cytochrome C Oxidase Assembly Factors Research Articles

More than Just Bread and Wine: Using Yeast to Understand Inherited Cytochrome Oxidase Deficiencies in Humans.

Inherited defects in cytochrome c oxidase (COX) are associated with a substantial subset of diseases adversely affecting the structure and function of the mitochondrial respiratory chain. This multi-subunit enzyme consists of 14 subunits and numerous cofactors, and it requires the function of some 30 proteins to assemble. COX assembly was first shown to be the primary defect in the majority of COX deficiencies 36 years ago. Over the last three decades, most COX assembly genes have been identified in the yeast Saccharomyces cerevisiae, and studies in yeast have proven instrumental in testing the impact of mutations identified in patients with a specific COX deficiency. The advent of accessible genome-wide sequencing capabilities has led to more patient mutations being identified, with the subsequent identification of several new COX assembly factors. However, the lack of genotype-phenotype correlations and the large number of genes involved in generating a functional COX mean that functional studies must be undertaken to assign a genetic variant as being causal. In this review, we provide a brief overview of the use of yeast as a model system and briefly compare the COX assembly process in yeast and humans. We focus primarily on the studies in yeast that have allowed us to both identify new COX assembly factors and to demonstrate the pathogenicity of a subset of the mutations that have been identified in patients with inherited defects in COX. We conclude with an overview of the areas in which studies in yeast are likely to continue to contribute to progress in understanding disease arising from inherited COX deficiencies.

Emerging mechanisms in the redox regulation of mitochondrial cytochrome c oxidase assembly and function.

In eukaryotic cells, mitochondria perform cellular respiration through a series of redox reactions ultimately reducing molecular oxygen to water. The system responsible for this process is the respiratory chain or electron transport system (ETS) composed of complexes I-IV. Due to its function, the ETS is the main source of reactive oxygen species (ROS), generating them on both sides of the mitochondrial inner membrane, i.e. the intermembrane space (IMS) and the matrix. A correct balance between ROS generation and scavenging is important for keeping the cellular redox homeostasis and other important aspects of cellular physiology. However, ROS generated in the mitochondria are important signaling molecules regulating mitochondrial biogenesis and function. The IMS contains a large number of redox sensing proteins, containing specific Cys-rich domains, that are involved in ETS complex biogenesis. The large majority of these proteins function as cytochrome c oxidase (COX) assembly factors, mainly for the handling of copper ions necessary for the formation of the redox reactive catalytic centers. A particular case of ROS-regulated COX assembly factor is COA8, whose intramitochondrial levels are increased by oxidative stress, promoting COX assembly and/or protecting the enzyme from oxidative damage. In this review, we will discuss the current knowledge concerning the role played by ROS in regulating mitochondrial activity and biogenesis, focusing on the COX enzyme and with a special emphasis on the functional role exerted by the redox sensitive Cys residues contained in the COX assembly factors.

145 President Oral Presentation Pick: Prenatal stress increases skeletal muscle mitochondrial volume density and function in yearling Brahman calves

Abstract We previously determined that prenatal stress (PNS) differentially affected methylation of DNA from leukocytes of 28-d-old calves. Specifically, COX14 (cytochrome c oxidase (COX) assembly factor) and CKMT1B (mitochondrial creatine kinase U-type) were hypomethylated and COA5 (COX assembly factor 5), COX5A (COX subunit 5A), NRF1 (nuclear respiratory factor 1), and GSST1 (glutathione S-transferase theta-1) were hypermethylated in PNS compared to non-PNS calves (P ≤ 0.05). Our current objective was to test the hypothesis that PNS exhibit impaired mitochondrial function and greater oxidative stress than non-PNS calves. Blood and longissimus dorsi muscle samples were collected from yearling Brahman calves whose mothers were stressed by 2 h transportation at 60, 80, 100, 120, and 140 days of gestation (PNS; 8 bulls, 6 heifers) and non-PNS calves (4 bulls, 6 heifers). Serum was evaluated for the stress hormone, cortisol, and muscle damage marker, creatine kinase; muscle was analyzed for mitochondrial volume density and function by citrate synthase (CS) and COX activities, respectively, concentration of malondialdehyde, a lipid peroxidation marker, and activity of the antioxidant, superoxide dismutase (SOD). Data were analyzed using mixed linear models with treatment and sex as fixed effects. Serum cortisol was numerically higher in PNS than non-PNS calves but was not statistically different. Muscle CS and COX activities relative to protein were greater in PNS than non-PNS calves (P ≤ 0.03), but COX relative to CS activity was similar between groups. Activity of COX was greater in bulls than heifers (P = 0.03), but no other measure was affected by sex. All other measures were unaffected by PNS. Prenatal stress did not affect markers of muscle damage and oxidative stress in yearling Brahman calves at rest but mitochondrial volume density and function were greater in PNS calves. Acute stressors induce oxidative stress, so implications of differences in mitochondria in PNS calves following a stressor should be investigated.

Consequences of Cytochrome c Oxidase Assembly Defects for the Yeast Stationary Phase

Functional assembly of cytochrome c oxidase (COX) is essential for an intact mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase remain largely unexplored. Stationary phase cultures are a good model for terminally differentiated cells in humans, such as neurons. Our goal is to define the physiological changes in mitochondrial respiration and oxidative stress between exponential and stationary phases in a respiratory competent strain and a set of COX assembly mutants with defects in a variety of aspects related to enzyme assembly. These results further our understanding of mitochondrial changes in terminally differentiated cells and aging cells. 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, which is supported by a decrease in protein levels of several COX assembly factors. Wild‐type cells also have a disrupted mitochondrial reticulum during stationary phase. Loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which we show 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. Specifically, differences in distribution of Sod1 between exponential and stationary phases give insight on location of reactive oxygen species (ROS) in the cell. Our results suggest that mitochondria may not be a major source of ROS at stationary phase in cells lacking an intact respiratory chain. Overall, we have identified fundamental differences between exponential and stationary phase mitochondria in respiratory competent yeast and have defined two distinct phenotypes, decreased levels of mitochondrial proteins and less mitochondrial oxidative damage, in cells with a defective respiratory chain. These findings suggest that COX deficiencies are associated with broader cellular phenotypes in terminally differentiated cells in humans.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase

The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits,together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

The Assembly Factor Pet117 Couples Heme a Synthase Activity to Cytochrome Oxidase Assembly

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for Cox15 oligomerization and function. Pet117 is shown to reside in the mitochondrial matrix, where it is associated with the inner membrane. Pet117 functions at the later maturation stages of the core CcO subunit Cox1 that precede Cox1 hemylation. Pet117 also physically interacts with Cox15 and specifically mediates the stability of Cox15 oligomeric complexes. This Cox15-Pet117 interaction observed by co-immunoprecipitation persists in the absence of heme a synthase activity, is dependent upon Cox1 synthesis and early maturation steps, and is further dependent upon the presence of the matrix-exposed, unstructured linker region of Cox15 needed for Cox15 oligomerization, suggesting that this region mediates the interaction or that the interaction is lost when Cox15 is unable to oligomerize. Based on these findings, it was concluded that Pet117 mediates coupling of heme a synthesis to the CcO assembly process in eukaryotes.

Human COA3 Is an Oligomeric Highly Flexible Protein in Solution.

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.

Cox1 mutation abrogates need for Cox23 in cytochrome c oxidase biogenesis.

Cox23 is a known conserved assembly factor for cytochrome c oxidase, although its role in cytochrome c oxidase (CcO) biogenesis remains unresolved. To gain additional insights into its role, we isolated spontaneous suppressors of the respiratory growth defect in cox23∆ yeast cells. We recovered independent colonies that propagated on glycerol/lactate medium for cox23∆ cells at 37°C. We mapped these mutations to the mitochondrial genome and specifically to COX1 yielding an I101F substitution. The I101F Cox1 allele is a gain-of-function mutation enabling yeast to respire in the absence of Cox23. CcO subunit steady-state levels were restored with the I101F Cox1 suppressor mutation and oxygen consumption and CcO activity were likewise restored. Cells harboring the mitochondrial genome encoding I101F Cox1 were used to delete genes for other CcO assembly factors to test the specificity of the Cox1 mutation as a suppressor of cox23∆ cells. The Cox1 mutant allele fails to support respiratory growth in yeast lacking Cox17, Cox19, Coa1, Coa2, Cox14 or Shy1, demonstrating its specific suppressor activity for cox23∆ cells.

Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model.

The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.

SCO2 Induces p53-Mediated Apoptosis by Thr845 Phosphorylation of ASK-1 and Dissociation of the ASK-1–Trx Complex

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

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.

COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.

Oligomerization of Heme o Synthase in Cytochrome Oxidase Biogenesis Is Mediated by Cytochrome Oxidase Assembly Factor Coa2

The synthesis of the heme a cofactor used in cytochrome c oxidase (CcO) is dependent on the sequential action of heme o synthase (Cox10) and heme a synthase (Cox15). The active state of Cox10 appears to be a homo-oligomeric complex, and formation of this complex is dependent on the newly synthesized CcO subunit Cox1 and the presence of an early Cox1 assembly intermediate. Cox10 multimerization is triggered by progression of Cox1 from the early assembly intermediate to downstream intermediates. The CcO assembly factor Coa2 appears important in coupling the presence of newly synthesized Cox1 to Cox10 oligomerization. Cells lacking Coa2 are impaired in Cox10 complex formation as well as the formation of a high mass Cox15 complex. Increasing Cox1 synthesis in coa2Δ cells restores respiratory function if Cox10 protein levels are elevated. The C-terminal segment of Cox1 is important in triggering Cox10 oligomerization. Expression of the C-terminal 54 residues of Cox1 appended to a heterologous matrix protein leads to efficient Cox10 complex formation in coa2Δ cells, but it fails to induce Cox15 complex formation. The state of Cox10 was evaluated in mutants, which predispose human patients to CcO deficiency and the neurological disorder Leigh syndrome. The presence of the D336V mutation in the yeast Cox10 backbone results in a catalytically inactive enzyme that is fully competent to oligomerize. Thus, Cox10 oligomerization and catalytic activation are separate processes and can be uncoupled.

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.

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.

Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.

Loss of function of Sco1 and its interaction with cytochromecoxidase

Sco1 and Sco2 are mitochondrial copper-binding proteins involved in the biogenesis of the Cu(A) site in the cytochrome c oxidase (CcO) subunit Cox2 and in the maintenance of cellular copper homeostasis. Human Surf1 is a CcO assembly factor with an important but poorly characterized role in CcO biogenesis. Here, we analyzed the impact on CcO assembly and tissue copper levels of a G132S mutation in the juxtamembrane region of SCO1 metallochaperone associated with early onset hypertrophic cardiomyopathy, encephalopathy, hypotonia, and hepatopathy, assessed the total copper content of various SURF1 and SCO2-deficient tissues, and investigated the possible physical association between CcO and Sco1. The steady-state level of mutant Sco1 was severely decreased in the muscle mitochondria of the SCO1 patient, indicating compromised stability and thus loss of function of the protein. Unlike the wild-type variant, residual mutant Sco1 appeared to migrate exclusively in the monomeric form on blue native gels. Both the activity and content of CcO were reduced in the patient's muscle to approximately 10-20% of control values. SCO1-deficient mitochondria showed accumulation of two Cox2 subcomplexes, suggesting that Sco1 is very likely responsible for a different posttranslational aspect of Cox2 maturation than Sco2. Intriguingly, the various SURF1-deficient samples analyzed showed a tissue-specific copper deficiency similar to that of SCO-deficient samples, suggesting a role for Surf1 in copper homeostasis regulation. Finally, both blue native immunoblot analysis and coimmunoprecipitation revealed that a fraction of Sco1 physically associates with the CcO complex in human muscle mitochondria, suggesting a possible direct relationship between CcO and the regulation of cellular copper homeostasis.

Transcriptional activators HAP/NF-Y rescue a cytochrome c oxidase defect in yeast and human cells

Cell survival and energy production requires a functional mitochondrial respiratory chain. Biogenesis of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a very complicated process and requires the assistance of a large number of accessory factors. Defects in COX assembly alter cellular respiration and produce severe human encephalomyopathies. Mutations in SURF1, a COX assembly factor of exact unknown function, produce Leigh's syndrome (LS), the most frequent cause of COX deficiency in infants. In the yeast Saccharomyces cerevisiae, deletion of the SURF1 homologue SHY1 results in a similar COX deficiency. In order to identify genetic modifiers of the shy1 mutant phenotype, we have explored for genetic interactions involving SHY1. Here we report that overexpression of Hap4p, the catalytic subunit of the CCAAT binding transcriptional activator Hap2/3/4/5p complex, suppresses the respiratory defect of yeast shy1 mutants by increasing the expression of nuclear-encoded COX subunits that interact with the mitochondrially encoded Cox1p. Analogously, overexpression of the Hap complex human homologue NF-YA/B/C transcription complex in SURF1-deficient fibroblasts from an LS patient efficiently rescues their COX deficiency.

Increased longevity and refractoriness to Ca2+-dependent neurodegeneration in Surf1 knockout mice

Leigh syndrome associated with cytochrome c oxidase (COX) deficiency is a mitochondrial disorder usually caused by mutations of SURF1, a gene encoding a putative COX assembly factor. We present here a Surf1-/- recombinant mouse obtained by inserting a loxP sequence in the open reading frame of the gene. The frequency of -/-, +/+ and +/- genotypes in newborn mice followed a mendelian distribution, indicating that the ablation of Surf1 is compatible with postnatal survival. The biochemical and assembly COX defect was present in Surf1(loxP)-/- mice, but milder than in humans. Surprisingly, not only these animals failed to show spontaneous neurodegeneration at any age, but they also displayed markedly prolonged lifespan, and complete protection from Ca(2+)-dependent neurotoxicity induced by kainic acid. Experiments on primary neuronal cultures showed markedly reduced rise of cytosolic and mitochondrial Ca(2+) in Surf1(loxP)-/- neurons, and reduced mortality, compared to controls. The mitochondrial membrane potential was unchanged in KO versus wild-type neurons, suggesting that the effects of the ablation of Surf1 on Ca(2+) homeostasis, and possibly on longevity, may be independent, at least in part, from those on COX assembly and mitochondrial bioenergetics.