The displacement of frataxin from the mitochondrial cristae correlates with abnormal respiratory supercomplexes formation and bioenergetic defects in cells of Friedreich ataxia patients.

Friedreich ataxia (FRDA) is a neurodegenerative disease resulting from a severe decrease of frataxin (FXN). Most patients carry a GAA repeat expansion in both alleles of the FXN gene, whereas a small fraction of them are compound heterozygous for the expansion and a point mutation in the other allele. FXN is involved in the mitochondrial biogenesis of the FeS-clusters. Distinctive feature of FRDA patient cells is an impaired cellular respiration, likely due to a deficit of key redox cofactors working as electrons shuttles through the respiratory chain. However, a definite relationship between FXN levels, FeS-clusters assembly dysregulation and bioenergetics failure has not been established. In this work, we performed a comparative analysis of the mitochondrial phenotype of cell lines from FRDA patients, either homozygous for the expansion or compound heterozygotes for the G130V mutation. We found that, in healthy cells, FXN and two key proteins of the FeS-cluster assembly machinery are enriched in mitochondrial cristae, the dynamic subcompartment housing the respiratory chain. On the contrary, FXN widely redistributes to the matrix in FRDA cells with defects in respiratory supercomplexes assembly and altered respiratory function. We propose that this could be relevant for the early mitochondrial defects afflicting FRDA cells and that perturbation of mitochondrial morphodynamics could in turn be critical in terms of disease mechanisms.

A-kinase-anchoring protein 1 (dAKAP1)-based signaling complexes coordinate local protein synthesis at the mitochondrial surface.

Compartmentalization of macromolecules is a ubiquitous molecular mechanism that drives numerous cellular functions. The appropriate organization of enzymes in space and time enables the precise transmission and integration of intracellular signals. Molecular scaffolds constrain signaling enzymes to influence the regional modulation of these physiological processes. Mitochondrial targeting of protein kinases and protein phosphatases provides a means to locally control the phosphorylation status and action of proteins on the surface of this organelle. Dual-specificity protein kinase A anchoring protein 1 (dAKAP1) is a multivalent binding protein that targets protein kinase A (PKA), RNAs, and other signaling enzymes to the outer mitochondrial membrane. Many AKAPs recruit a diverse set of binding partners that coordinate a broad range of cellular processes. Here, results of MS and biochemical analyses reveal that dAKAP1 anchors additional components, including the ribonucleoprotein granule components La-related protein 4 (LARP4) and polyadenylate-binding protein 1 (PABPC1). Local translation of mRNAs at organelles is a means to spatially control the synthesis of proteins. RNA-Seq data demonstrate that dAKAP1 binds mRNAs encoding proteins required for mitochondrial metabolism, including succinate dehydrogenase. Functional studies suggest that the loss of dAKAP1-RNA interactions reduces mitochondrial electron transport chain activity. Hence, dAKAP1 plays a previously unappreciated role as a molecular interface between second messenger signaling and local protein synthesis machinery.

Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophila.

Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. However, the underlying mechanism for this dynamic regulation remains unclear. Here, we show that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. We further reveal that transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. Our study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development.

Obesity and steatosis promotes mitochondrial remodeling that enhances respiratory capacity in the liver of ob/ob mice.

We investigated if obesity/steatosis promotes mitochondrial remodeling in the liver of ob/ob mice (an obesity model). Liver mitochondria from ob/ob mice (21 weeks with significant steatosis) had ~ 2-fold increases in state III respiration compared with control (C57BL/6J, C57BL/6NJ) for all respiratory substrates examined (glutamate/malate, succinate, octanoate, and glycerol 3-phosphate). A corresponding 2-fold increase in the expression of respiratory complexes (I, IV, and V) and other respiratory proteins (glycerol phosphate dehydrogenase-2 and medium-chain acyl-coenzyme A dehydrogenase) occur in liver mitochondria of mature ob/ob mice. Conversely, respiration in liver mitochondria from young ob/ob mice (6 weeks) does not differ from control with any respiratory substrates examined. Overall, mitochondrial remodeling that enhances respiration increases with obesity/steatosis in the liver of ob/ob mice.

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.

Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site.

Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

CEP-1, the Caenorhabditis elegans p53 homolog, mediates opposing longevity outcomes in mitochondrial electron transport chain mutants.

Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the "aging" Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection.

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport.

Downregulation of the expression of mitochondrial electron transport complex genes in autism brains.

Mitochondrial dysfunction (MtD) and abnormal brain bioenergetics have been implicated in autism, suggesting possible candidate genes in the electron transport chain (ETC). We compared the expression of 84 ETC genes in the post-mortem brains of autism patients and controls. Brain tissues from the anterior cingulate gyrus, motor cortex, and thalamus of autism patients (n = 8) and controls (n = 10) were obtained from Autism Tissue Program, USA. Quantitative real-time PCR arrays were used to quantify gene expression. We observed reduced expression of several ETC genes in autism brains compared to controls. Eleven genes of Complex I, five genes each of Complex III and Complex IV, and seven genes of Complex V showed brain region-specific reduced expression in autism. ATP5A1 (Complex V), ATP5G3 (Complex V) and NDUFA5 (Complex I) showed consistently reduced expression in all the brain regions of autism patients. Upon silencing ATP5A1, the expression of mitogen-activated protein kinase 13 (MAPK13), a p38 MAPK responsive to stress stimuli, was upregulated in HEK 293 cells. This could have been induced by oxidative stress due to impaired ATP synthesis. We report new candidate genes involved in abnormal brain bioenergetics in autism, supporting the hypothesis that mitochondria, critical for neurodevelopment, may play a role in autism.

Toxicity of neurons treated with herbicides and neuroprotection by mitochondria-targeted antioxidant SS31.

The purpose of this study was to determine the neurotoxicity of two commonly used herbicides: picloram and triclopyr and the neuroprotective effects of the mitochondria-targeted antioxidant, SS31. Using mouse neuroblastoma (N2a) cells and primary neurons from C57BL/6 mice, we investigated the toxicity of these herbicides, and protective effects of SS1 peptide against picloram and triclopyr toxicity. We measured total RNA content, cell viability and mRNA expression of peroxiredoxins, neuroprotective genes, mitochondrial-encoded electron transport chain (ETC) genes in N2a cells treated with herbicides and SS31. Using primary neurons from C57BL/6 mice, neuronal survival was studied in neurons treated with herbicides, in neurons pretreated with SS31 plus treated with herbicides, neurons treated with SS31 alone, and untreated neurons. Significantly decreased total RNA content, and cell viability in N2a cells treated with picloram and triclopyr were found compared to untreated N2a cells. Decreased mRNA expression of neuroprotective genes, and ETC genes in cells treated with herbicides was found compared to untreated cells. Decreased mRNA expression of peroxiredoxins 1-6 in N2a cells treated with picloram was found, suggesting that picloram affects the antioxidant enzymes in N2a cells. Immunofluorescence analysis of primary neurons revealed that decreased neuronal branching and degenerating neurons in neurons treated with picloram and triclopyr. However, neurons pretreated with SS31 prevented degenerative process caused by herbicides. Based on these results, we propose that herbicides--picloram and triclopyr appear to damage neurons, and the SS31 peptide appears to protect neurons from herbicide toxicity.

Abnormal mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in Huntington's disease: implications for selective neuronal damage.

The purpose of our study was to determine the relationship between mutant huntingtin (Htt) and mitochondrial dynamics in the progression of Huntington's disease (HD). We measured the mRNA levels of electron transport chain genes, and mitochondrial structural genes, Drp1 (dynamin-related protein 1), Fis1 (fission 1), Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), Opa1 (optric atrophy 1), Tomm40 (translocase of outermembrane 40) and CypD (cyclophilin D) in grade III and grade IV HD patients and controls. The mutant Htt oligomers and the mitochondrial structural proteins were quantified in the striatum and frontal cortex of HD patients. Changes in expressions of the electron transport chain genes were found in HD patients and may represent a compensatory response to mitochondrial damage caused by mutant Htt. Increased expression of Drp1 and Fis1 and decreased expression of Mfn1, Mfn2, Opa1 and Tomm40 were found in HD patients relative to the controls. CypD was upregulated in HD patients, and this upregulation increased as HD progressed. Significantly increased immunoreactivity of 8-hydroxy-guanosine was found in the cortical specimens from stage III and IV HD patients relative to controls, suggesting increased oxidative DNA damage in HD patients. In contrast, significantly decreased immunoreactivities of cytochrome oxidase 1 and cytochrome b were found in HD patients relative to controls, indicating a loss of mitochondrial function in HD patients. Immunoblotting analysis revealed 15, 25 and 50 kDa mutant Htt oligomers in the brain specimens of HD patients. All oligomeric forms of mutant Htt were significantly increased in the cortical tissues of HD patients, and mutant Htt oligomers were found in the nucleus and in mitochondria. The increase in Drp1, Fis1 and CypD and the decrease in Mfn1 and Mfn2 may be responsible for abnormal mitochondrial dynamics that we found in the cortex of HD patients, and may contribute to neuronal damage in HD patients. The presence of mutant Htt oligomers in the nucleus of HD neurons and in mitochondria may disrupt neuronal functions. Based on these findings, we propose that mutant Htt in association with mitochondria imbalance and mitochondrial dynamics impairs axonal transport of mitochondria, decreases mitochondrial function and damages neurons in affected brain regions of HD patients.

Effects of TCDD on the expression of nuclear encoded mitochondrial genes.

Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 nuclear genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 microg/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 h) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (/fold change/ > 1.5 and P-value < 0.1). Of these, 8 exhibited a sigmoidal or exponential dose-response profile (0.03 to 300 microg/kg TCDD) at 4, 24 or 72 h. Dose-responsive genes encoded proteins associated with electron transport chain (ETC) complexes I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of all 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity.

Impact of molecular hydrogen on chalcopyrite bioleaching by the extremely thermoacidophilic archaeon Metallosphaera sedula.

Hydrogen served as a competitive inorganic energy source, impacting the CuFeS(2) bioleaching efficiency of the extremely thermoacidophilic archaeon Metallosphaera sedula. Open reading frames encoding key terminal oxidase and electron transport chain components were triggered by CuFeS(2). Evidence of heterotrophic metabolism was noted after extended periods of bioleaching, presumably related to cell lysis.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non-replicating persistence.

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Response of Saccharomyces cerevisiae to stress-free acidification.

Genome-wide transcriptional analysis of a Saccharomyces cerevisiae batch culture revealed that more than 829 genes were regulated in response to an environmental shift from pH 6 to pH 3 by added sulfuric acid. This shift in pH was not detrimental to the rate of growth compared to a control culture that was maintained at pH 6 and the transcriptional changes most strikingly implicated not up- but down-regulation of stress responses. In addition, the transcriptional changes upon acid addition indicated remodeling of the cell wall and central carbon metabolism. The overall trend of changes was similar for the pH-shift experiment and the pH 6 control. However, the changes in the pH 6 control were much weaker and occurred 2.5 h later than in the pH-shift experiment. Thus, the reaction to the steep pH decrease was an immediate response within the normal repertoire of adaptation shown in later stages of fermentation at pH 6. Artificially preventing the yeast from acidifying the medium may be considered physiologically stressful under the tested conditions.

Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes.

The crenarchaeal order Sulfolobales collectively contain at least five major terminal oxidase complexes. Based on genome sequence information, all five complexes are found only in Metallosphaera sedula and Sulfolobus tokodaii, the two sequenced Sulfolobales capable of iron oxidization. While specific respiratory complexes in certain Sulfolobales have been characterized previously as proton pumps for maintaining intracellular pH and generating proton motive force, their contribution to sulfur and iron biooxidation has not been considered. For M. sedula growing in the presence of ferrous iron and reduced inorganic sulfur compounds (RISCs), global transcriptional analysis was used to track the response of specific genes associated with these complexes, as well as other known and putative respiratory electron transport chain elements. Open reading frames from all five terminal oxidase or bc(1)-like complexes were stimulated on one or more conditions tested. Components of the fox (Msed0467 to Msed0489) and soxNL-cbsABA (Msed0500 to Msed0505) terminal/quinol oxidase clusters were triggered by ferrous iron, while the soxABCDD' terminal oxidase cluster (Msed0285 to Msed0291) were induced by tetrathionate and S(0). Chemolithotrophic electron transport elements, including a putative tetrathionate hydrolase (Msed0804), a novel polysulfide/sulfur/dimethyl sulfoxide reductase-like complex (Msed0812 to Msed0818), and a novel heterodisulfide reductase-like complex (Msed1542 to Msed1550), were also stimulated by RISCs. Furthermore, several hypothetical proteins were found to have strong responses to ferrous iron or RISCs, suggesting additional candidates in iron or sulfur oxidation-related pathways. From this analysis, a comprehensive model for electron transport in M. sedula could be proposed as the basis for examining specific details of iron and sulfur oxidation in this bioleaching archaeon.

Calcium-dependent up-regulation of mitochondrial electron transfer chain gene expressions in human luteinized granulosa cells.

OBJECTIVE: To evaluate the transcription and translation ability of mitochondria in terminally differentiated granulosa cells, these cells were incubated with ionic calcium. DESIGN: Prospective laboratory research. SETTING: In vitro fertilization laboratory in a university hospital. PATIENT(S): Granulosa cells were harvested from 50 female patients undergoing IVF. INTERVENTION(S): Analysis of mitochondrial gene expression by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and of mitochondrial-encoded proteins by Western blot. MAIN OUTCOME MEASURE(S): Comparison of the RNA expression levels of genes including cytochrome c oxidase subunit I (COX I), adenosine triphosphate synthase 6 (ATPase 6), flavoprotein, and succinate-ubiquinone oxidoreductase, and protein levels of COX I and flavoprotein in different calcium ion treatment groups. RESULT(S): There were dose-dependent increases in RNA expressions of the four genes analyzed from granulosa cells cultured in a serial concentration of calcium ions. This effect was abolished when cells were preincubated with the extracellular calcium-chelating agent, Ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The effect of ionic calcium on both the nuclear- and mitochondrial-encoded subunits also was determined. Expression levels of mitochondrial transcription factor A in RNA significantly increased in granulosa cells that were exposed for 24 and 48 hours to 0.5 and 1 microM A23187. CONCLUSION(S): The present study is the first report to present calcium-dependent increases in the transcription and translation levels of both nuclear-encoded and mitochondrial-encoded mitochondrial respiratory enzyme subunits and also indicates that mitochondrial transcription factor A is involved in mitochondrial biogenesis.