Peralta S.,Autonomous University of Madrid |
Peralta S.,University of Miami |
Clemente P.,Autonomous University of Madrid |
Sanchez-Martinez A.,Autonomous University of Madrid |
And 9 more authors.
Journal of Biological Chemistry | Year: 2012
In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5′-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56D6 and ccdc56D11 alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. Wepropose CCDC56 as a candidate factor required for COX biogenesis/assembly. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Echevarria L.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC |
Echevarria L.,Institute Investigacion Sanitaria 12 Of Octubre I12 |
Clemente P.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC |
Clemente P.,Institute Investigacion Sanitaria 12 Of Octubre I12 |
And 7 more authors.
Biochemical Journal | Year: 2014
Translational accuracy depends on the correct formation of aminoacyl-tRNAs, which, in the majority of cases, are produced by specific aminoacyl-tRNA synthetases that ligate each amino acid to its cognate isoaceptor tRNA. Aminoacylation of tRNAGln, however, is performed by various mechanisms in different systems. Since no mitochondrial glutaminyl-tRNA synthetase has been identified to date in mammalian mitochondria, GlntRNAGln has to be formed by an indirect mechanism in the organelle. It has been demonstrated that human mitochondria contain a non-discriminating glutamyl-tRNA synthetase and the heterotrimeric enzyme GatCAB (where Gat is glutamyltRNAGln amidotransferase), which are able to catalyse the formation of Gln-tRNAGln in vitro. In the present paper we demonstrate that mgatA (mouse GatA) interference inmouse cells produces a strong defect in mitochondrial translation without affecting the stability of the newly synthesized proteins. As a result, interfered cells present an impairment of the oxidative phosphorylation system and a significant increase in ROS (reactive oxygen species) levels. MS analysis of mitochondrial proteins revealed no glutamic acid found in the position of glutamines, strongly suggesting that misaminoacylated GlutRNAGln is rejected from the translational apparatus to maintain the fidelity of mitochondrial protein synthesis in mammals. © 2014 Biochemical Society.