RESUMEN
Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Uranio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Compuestos Férricos/metabolismo , Membrana Celular/metabolismo , Cationes/química , Cationes/metabolismo , Uranio/química , Proteínas de Unión al Calcio/metabolismoRESUMEN
Uranium (U) is a naturally occurring radionuclide that is toxic to plants. It is known to interfere with phosphate nutrition and to modify the expression of iron (Fe)-responsive genes. The transporters involved in the uptake of U from the environment are unknown. Here, we addressed whether IRT1, a high-affinity Fe2+ transporter, could contribute to U uptake in Arabidopsis thaliana. An irt1 null mutant was grown hydroponically in different conditions of Fe bioavailability and phosphate supply, and challenged with uranyl. Several physiological parameters (fitness, photosynthesis) were measured to evaluate the response to U treatment. We found that IRT1 is not a major route for U uptake in our experimental conditions. However, the analysis of irt1 indicated that uranyl interferes with Fe and phosphate homeostasis at different levels. In phosphate-sufficient conditions, the absence of the cation chelator EDTA in the medium has drastic consequences on the physiology of irt1, with important symptoms of Fe deficiency in chloroplasts. These effects are counterbalanced by U, probably because the radionuclide competes with Fe for complexation with phosphate and thus releases active Fe for metabolic and biogenic processes. Our study reveals that challenging plants with U is useful to decipher the complex interplay between Fe and phosphate.