RESUMEN
Bacterial GatCAB amidotransferases are responsible for the transamidation of mischarged glutamyl-tRNA(Gln) into glutaminyl-tRNA(Gln). Mitochondria matrix also has a multienzymatic complex necessary for the transamidation of glutamyl-tRNA(Gln). Gtf1p, Her2p and Pet112p are the constituents of mitochondrial GatFAB amidotransferase complex. Her2p is subunit A of GatFAB complex, while Gtf1p is subunit F, a connector protein between Pet112p (subunit B) and Her2p. Here we evaluate through molecular modeling and amino acid correlation analysis the HER2 protein family. Localization studies indicated that Her2p is predominantly localized in the mitochondrial outer membrane, but it is also located in the mitochondrial matrix where together with Pet112p and Gtf1p constitutes the GatFAB complex. Finally, HER2 random mutagenesis unveiled important residues that provide thermo stability for the complex and are differently suppressed by overexpression of GTF1 or PET112. For instance, her2/ts11 mutant showed its fermentative growth impaired, and poorly rescued by GTF1 indicating that Her2p unknown function in the mitochondria outer membrane affects cell viability.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Supervivencia Celular , Mapeo Cromosómico , Retículo Endoplásmico/metabolismo , Glutamina/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Mutación , Aminoacil-ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Transaminasas/metabolismoRESUMEN
The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.