The plasma membrane-associated cation-binding protein PCaP1 of Arabidopsis thaliana is a uranyl-binding protein.

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.

Development of a metalloproteomic approach to analyse the response of Arabidopsis cells to uranium stress.

Uranium is a naturally occurring radionuclide that is absorbed by plants and interferes with many aspects of their physiology and development. In this study, we used an ionomic, metalloproteomic, and biochemical approach to gain insights into the impact of uranyl ions on the proteome of Arabidopsis thaliana cells. First, we showed that most of the U was trapped in the cell wall and only a small amount of the radionuclide was found in the cell-soluble fraction. Also, the homeostasis of several essential elements was significantly modified in the cells challenged with U. Second, the soluble proteome from Arabidopsis cells was fractionated into 10 subproteomes using anion-exchange chromatography. Proteomic analyses identified 3676 proteins in the different subproteomes and the metal-binding proteins were profiled using inductively coupled plasma mass spectrometry. Uranium was detected in several chromatographic fractions, indicating for the first time that several pools of Arabidopsis proteins are capable of binding the uranyl ion in vivo. Third, we showed that the pattern of some lysine and arginine methylated proteins was modified following exposure to U. We further identified that the ribosomal protein RPS10C was dimethylated at two arginine residues in response to uranyl ion stress. Together, these results provide the first clues for the impact of U on the Arabidopsis proteome and pave the way for the future identification of U-binding proteins.