RESUMO
We earlier reported 'PeIF5B' as a novel factor from Pisum sativum that has sequence similarity to eIF5B (S. Rasheedi, S. Ghosh, M. Suragani et al., P. sativum contains a factor with strong homology to eIF5B, Gene 399 (2007) 144-151). The main aim of the present study was to perform functional characterization of PeIF5B as an eIF5B homologue from plant system. PeIF5B shows binding to Met - tRNA(f)(Met), hydrolyses GTP and interacts with ribosomes. In vivo growth complementation analysis shows that PeIF5B partially complements its yeast homologue. Interestingly, PeIF5B mainly localizes in the nucleus as confirmed by nuclear localization signal (NLS) prediction, confocal imaging and immunoblots of cellular fractions. Similar to the yeast eIF5B but unlike the human orthologue, PeIF5B is an intron-less gene. This study highlights PeIF5B's role as a functional eIF5B homologue possibly participating in nuclear translation in plant system.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Genes de Plantas/genética , Pisum sativum/genética , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas/genética , Fatores de Iniciação em Eucariotos/genética , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Confocal , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Cellular iron levels are closely monitored by iron regulatory and sensor proteins of Mycobacterium tuberculosis for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of M. tuberculosis aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that M. tuberculosis Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to cis-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays, M. tuberculosis Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3' UTR of thioredoxin (trxC) and the 5' UTR of the iron-dependent repressor and activator (ideR) of M. tuberculosis. M. tuberculosis Acn when reactivated with Fe(2+) functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of M. tuberculosis Acn are mutually exclusive. Our results demonstrate the bifunctional nature of M. tuberculosis Acn, pointing to its likely role in iron homeostasis.
