Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co-ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co-ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear-encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N-terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear-encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion.     

Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise

Mitochondrial dysfunction, associated with insulin resistance, is characterized by low expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and nuclear-encoded mitochondrial genes. This deficit could be due to decreased physical activity or a decreased response of gene expression to exercise. The objective of this study was to investigate whether a bout of exercise induces the same increase in nuclear-encoded mitochondrial gene expression in insulin-sensitive and insulin-resistant subjects matched for exercise capacity. Seven lean and nine obese subjects took part. Insulin sensitivity was assessed by an 80 mU.m(-2).min(-1) euglycemic clamp. Subjects were matched for aerobic capacity and underwent a single bout of exercise at 70 and 90% of maximum heart rate with muscle biopsies at 30 and 300 min postexercise. Quantitative RT-PCR and immunoblot analyses were used to determine the effect of exercise on gene expression and protein abundance and phosphorylation. In the postexercise period, lean subjects immediately increased PGC-1alpha mRNA level (reaching an eightfold increase by 300 min postexercise) and protein abundance and AMP-dependent protein kinase phosphorylation. Activation of PGC-1alpha was followed by increase of nuclear respiratory factor-1 and cytochrome c oxidase (subunit VIc). However, in insulin-resistant subjects, there was a delayed and reduced response in PGC-1alpha mRNA and protein, and phosphorylation of AMP-dependent protein kinase was transient. None of the genes downstream of PGC-1alpha was increased after exercise in insulin resistance. Insulin-resistant subjects have a reduced response of nuclear-encoded mitochondrial genes to exercise, and this could contribute to the origin and maintenance of mitochondrial dysfunction.     

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.     

Nuclear-encoded subunits of human cytochrome c oxidase: SstI restriction fragment length polymorphism

A DNA polymorphism of the nuclear-encoded subunit Va of the human cytochrome c oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was detected in genes for the subunits IV and Vb of the same enzyme.