Search

Aims: Mitochondrial cytochrome c oxidase (COX) subunit 5 and cytochrome c (Cyc) exist in two isoforms, transcriptionally regulated by oxygen in yeast. The gene pair COX5a/CYC1 encodes the normoxic isoforms (Cox5a and iso1-Cyc) and the gene pair COX5b/CYC7 encodes the hypoxic isoforms (Cox5b and iso2-Cyc). Rox1 is a transcriptional repressor of COX5b/CYC7 in normoxia. COX5b is additionally repressed by Ord1. Here, we investigated whether these pathways respond to environmental and mitochondria-generated oxidative stress., Results: The superoxide inducer menadione triggered a significant de-repression of COX5b and CYC7. Hydrogen peroxide elicited milder de-repression effects that were enhanced in the absence of Yap1, a key determinant in oxidative stress resistance. COX5b/CYC7 was also de-repressed in wild-type cells treated with antimycin A, a mitochondrial bc1 complex inhibitor that increases superoxide production. Exposure to menadione and H2O2 enhanced both, Hap1-independent expression of ROX1 and Rox1 steady-state levels without affecting Ord1. However, oxidative stress lowered the occupancy of Rox1 on COX5b and CYC7 promoters, thus inducing their de-repression., Innovation: Reactive oxygen species (ROS)-induced hypoxic gene expression in normoxia involves the oxygen-responding Rox1 transcriptional machinery. Contrary to what occurs in hypoxia, ROS enhances Rox1 accumulation. However, its transcriptional repression capacity is compromised., Conclusion: ROS induce expression of hypoxic COX5b and CYC7 genes through an Ord1- and Hap1-independent mechanism that promotes the release of Rox1 from or limits the access of Rox1 to its hypoxic gene promoter targets.

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, the assays describe methods that form a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues.

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been progressively developed. Based on methods described elsewhere, the use of blue native gel electrophoresis (BNGE) techniques to routinely assess the OXPHOS system and screen for enzymatic defects in homogenates or mitochondrial preparations from tissues or cultured cells is described here.

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. Several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, this unit describes the creation and study of yeast models of mitochondrial OXPHOS deficiencies.

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.

Mitochondrial dysfunction may play an important role in the pathogenic mechanism of Huntington's disease (HD). However, the exact mechanism by which mutated huntingtin could cause bioenergetic dysfunction is still unknown. We have constructed a stable inducible yeast model of HD by expressing a human huntingtin fragment containing a mutant polyglutamine tract of 103Q fused to green fluorescent protein (GFP), and a control expressing a wild-type 25Q domain fused to GFP in a wild-type strain. We showed that in yeast cells expressing 103Q, cell respiration was progressively reduced after 4-6 h of induction with galactose, down to 50% of the control after 10 h of induction. The cell respiration defect results from an alteration in the function and amount of mitochondrial respiratory chain complex II+III, in congruency to data obtained from postmortem brain of HD patients and from toxin models. In our model, the production of reactive oxygen species (ROS) is significantly enhanced in cells expressing 103Q. Quenching of ROS with resveratrol partially prevents the cell respiration defect. Mitochondrial morphology and distribution were also altered in cells expressing 103Q, probably resulting from the interaction of aggregates with portions of the mitochondrial web and from a progressive disruption of the actin cytoskeleton. We propose a mechanism for mitochondrial dysfunction in our yeast model of HD in which the interactions of misfolded/aggregated polyglutamine domains with the mitochondrial and actin networks lead to disturbances in mitochondrial distribution and function and to increase in ROS production. Oxidative damage could preferentially affect the stability and function of enzymes containing iron-sulfur clusters such as complexes II and III. Our yeast model represents a very useful paradigm to study mitochondrial physiology alterations in the pathogenic mechanism of HD.

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (ρ° cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming ρ° cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndil and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of ρ° cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD[sup+]. Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes. [ABSTRACT FROM AUTHOR]

Objective: To study the mitochondrial respiratory chain enzyme activities in patients with idiopathic dilated cardiomyopathy (IDC). Methods: Mitochondrial respiratory chain enzyme activities were assessed spectrophotometrically in left ventricular tissue of 17 patients with IDC undergoing cardiac transplantation, as well as in two groups of controls: a group of six patients suffering from ischemic dilated cardiomyopathy (IC) also undergoing cardiac transplantation, and a group of 17 organ donors considered normal from a cardiac point of view. Cytochrome b gene from three IDC patients whose complex III activity was particularly low and from three controls was also sequenced. Results: We found that complex III enzymatic activity was lower not only in IDC but also in IC patients when compared with normal controls. When analysing cytochrome b gene we only found neutral polymorphisms previously described. Conclusions: In view of such results, we believe that the decrease of respiratory chain complex III activity found in some cases of IDC is a secondary phenomenon, and not due to a primary mitochondrial disease. [ABSTRACT FROM PUBLISHER]

Human immunodeficiency virus type I (HIV-1) exploits various host cellular pathways for efficient infection. Here we report that the absence of mitochondrial DNA (mtDNA) in ρ0 cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ0 cells is not simply the result of impaired oxidative phosphorylation (OXPHOS) because pharmacological OXPHOS inhibition did not inhibit HIV-1 infection. Analysis of the early steps of virus infection by real-time PCR quantification of stage-specific HIV-1 DNA products in the infected ρ0 and parental cell line have allowed us to conclude that HIV-1 infection in ρ0 cells is blocked at the steps that occur after reverse transcription and prior to nuclear import. Additionally, confocal fluorescence microscope analysis showed that the majority of viral complexes containing HIV-1 p24 co-localize with mitochondria in target cells, suggesting an interaction between the two. Collectively, our data strongly indicate that mitochondria play an important role during early stages of HIV-1 infection, probably through direct association with HIV-1 intracellular complexes. [ABSTRACT FROM AUTHOR]