title: Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium.
abstract: Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Saclay Plant Sciences, Université Paris-Saclay, CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, F-91198, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Pottier
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Saclay Plant Sciences, Université Paris-Saclay, CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, F-91198, France.
authors.forename: Ronald
authors.initials: R
authors.lastname: Oomen
authors.affiliation: Istituto di Biofisica, CNR, Genova, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Picco
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Saclay Plant Sciences, Université Paris-Saclay, CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, F-91198, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Giraudat
authors.affiliation: Istituto di Biofisica, CNR, Genova, Italy.
authors.forename: Joachim
authors.initials: J
authors.lastname: Scholz-Starke
authors.affiliation: Laboratoire de Bioénergie et Biotechnologie des Bactéries et Microalgues, CEA, DSV, IBEB, Saint-Paul-lès-Durance, F-13108, France.|CNRS, UMR Biol Veget et Microbiol Environ, Saint-Paul-lès-Durance, F-13108, France.|Aix-Marseille Université, Saint-Paul-lès-Durance, F-13108, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Richaud
authors.affiliation: Istituto di Biofisica, CNR, Genova, Italy.
authors.forename: Armando
authors.initials: A
authors.lastname: Carpaneto
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), Saclay Plant Sciences, Université Paris-Saclay, CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, F-91198, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Thomine
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000633:toxicity*; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D008670:Metals / Q000378:metabolism; D009154:Mutation; D010940:Plant Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008670:Metals; D010940:Plant Proteins; D002104:Cadmium
keywords: Arabidopsis thaliana; transition metals; transport; vacuole