RESUMO
Assembly of cytochromes c is mediated by different proteins depending on the organism and organelle considered. In land plants, mitochondria follow a pathway distinct from that of yeast and animal mitochondria, more similar to that described for alpha- and gamma-proteobacteria. Indeed, in plant mitochondria, four genes were identified based on the similarities of their products with bacterial proteins involved in c-type cytochrome maturation. We report the characterisation of one of these mitochondrial genes in Triticum aestivum, TaccmB, which is proposed to encode a subunit of an ABC transporter. The transcript extremities were mapped and cDNA sequencing revealed 42 C to U editing positions in the 618 nucleotide long coding region. This high editing rate affects the identity of 32 amino acids out of 206. Antibodies directed against wheat CcmB recognise a 28 kDa protein in an enriched inner mitochondrial membrane protein fraction, a location which is in agreement with the high hydrophobicity of the protein and its function as a putative transmembrane domain of an ABC transporter involved in cytochrome c and c1 biogenesis in plant mitochondria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Grupo dos Citocromos c/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas de Plantas , Triticum/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Edição de RNA , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid beta-oxidation. In plants, fatty acid beta-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Mitocôndrias/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Etiquetas de Sequências Expressas , Genes de Plantas/genética , Isovaleril-CoA Desidrogenase , Cinética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica/genética , Oxirredutases/química , Oxirredutases/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The gene and cDNA of an Arabidopsis thaliana cytidine deaminase (CDA) were cloned and sequenced. The gene, At-cda1, is located on chromosome 2 and is expressed in all plant tissues tested, although with quantitative differences. Expression analysis suggest that At-cda1 probably codes for the housekeeping cytidine deaminase of Arabidopsis. The gene was functionally expressed in Escherichia coli and the protein, At-CDA1, shows similar enzymatic and substrate specificities as conventional cytidine deaminases: it deaminates cytidine and deoxycytidine and is competitively inhibited by cytosine-containing compounds. Because the protein shows no affinity to RNA, it is not likely to be involved in RNA-editing by C-to-U deamination. When compared to cytidine deaminases from other organisms, it becomes clear that At-CDA1 is related, both in sequence and structure, to the CDA of E. coli and other gram-negative bacteria. The eubacterial nature of the Arabidopsis CDA suggests that it is an additional example of a plant gene of endosymbiotic origin.
