Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

• Published in: Metallomics 2015-01, Vol.7 (4), p.691-701
• Main Authors: Albareda, Marta, Rodrigue, Agnès, Brito, Belén, Ruiz-Argüeso, Tomás, Imperial, Juan, Mandrand-Berthelot, Marie-Andrée, Palacios, Jose
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacteriology; Biochemistry; Biochemistry, Molecular Biology; Escherichia coli - metabolism; Genes, Reporter; Genomics; Hydrogenase - chemistry; Life Sciences; Ligands; Lipid Bilayers - chemistry; Membrane Proteins - chemistry; Microbiology and Parasitology; Molecular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nickel - chemistry; Protein Structure, Tertiary; Rhizobium leguminosarum - chemistry
• ISSN: 1756-5901; 1756-591X
• DOI: 10.1039/c4mt00298a

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ± 21 nM; Vmax 49 ± 21 pmol Ni(2+) min(-1) mg(-1) bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(ii) ions. This Ni(2+) transport is not inhibited by similar cations such as Mn(2+), Zn(2+), or Co(2+), but is blocked by Cu(2+). Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.