OBJECTIVE: Dictyostelium is a professional phagocyte, which grows on bacteria. Its genome harbours two Nramp genes: Nramp1, the ortholog of mammalian Nramp1, is expressed in phagosomes/macropinosomes and confers resistance against pathogenic bacteria; Nramp2 is localized in the contractile vacuole, and synergistically with Nramp1 regulates iron homeostasis. Objective of this study was to characterize the transport properties of both proteins. METHODS: Dictyostelium WT strain, Nramp1 and Nramp2-KO mutants were used. For assaying iron traffic in macropinosomes, cells were incubated with calcein and increasing amount of divalent metals, and assessed for calcein fluorescence de-quenching by confocal microscopy and flow cytometry. Functional characterization studies were done with Xenopus oocytes. For expression on oocyte surface, the N- and C-termini of Nramp genes were replaced with corresponding regions of murine DMT1. Following cRNA injection, oocytes were subjected to radiochemical and electrophysiological assays. The oocytes were also injected with calcein, incubated with divalent metals and calcein-quenching assessed in confocal microscopy. RESULTS: Calcein is ingested by macropinocytosis. We incubated wild-type and Nramp-null mutants with Fe2+- or Fe3+-quenched calcein. Calcein de-quenching occurred in macropinosomes of all strains, except Nramp1-null mutant, suggesting that Nramp1 is essential for iron export. Mn or Cu did not affect de-quencing, suggesting that their transport is Nramp1-independent. In calcein-loaded Xenopus oocytes expressing Nramp1, Fe2+ or Mn2+, not Fe3+, led to fluorescence-quenching. Cu2+ favoured iron transport inside-out. In electrophysiological studies, Fe2+, Cd and Co, but not Fe3+ or Cu, elicited inward currents, confirming that Nramp1 does not transport Fe3+ and Cu. Radiochemical studies showed that Fe2+ transport is proton-dependent, and competed by Mn, Cd, Ni and Co, to a lower extent by Zn and Cu. CONCLUSION: Dictyostelium Nramp1 is an electrogenic proton-dependent iron transporter with cation selectivity comparable to mammalian DMT1. It transports Fe2+, not Fe3+ and is essential for Fe2+ export from macropinosomes.
Functional characterization of Dictyostelium Nramp1 and Nramp2, which confer resistance to bacterial infection and are essential for iron transport
BURACCO, SIMONA;PERACINO, Barbara;BOZZARO, Salvatore
2014-01-01
Abstract
OBJECTIVE: Dictyostelium is a professional phagocyte, which grows on bacteria. Its genome harbours two Nramp genes: Nramp1, the ortholog of mammalian Nramp1, is expressed in phagosomes/macropinosomes and confers resistance against pathogenic bacteria; Nramp2 is localized in the contractile vacuole, and synergistically with Nramp1 regulates iron homeostasis. Objective of this study was to characterize the transport properties of both proteins. METHODS: Dictyostelium WT strain, Nramp1 and Nramp2-KO mutants were used. For assaying iron traffic in macropinosomes, cells were incubated with calcein and increasing amount of divalent metals, and assessed for calcein fluorescence de-quenching by confocal microscopy and flow cytometry. Functional characterization studies were done with Xenopus oocytes. For expression on oocyte surface, the N- and C-termini of Nramp genes were replaced with corresponding regions of murine DMT1. Following cRNA injection, oocytes were subjected to radiochemical and electrophysiological assays. The oocytes were also injected with calcein, incubated with divalent metals and calcein-quenching assessed in confocal microscopy. RESULTS: Calcein is ingested by macropinocytosis. We incubated wild-type and Nramp-null mutants with Fe2+- or Fe3+-quenched calcein. Calcein de-quenching occurred in macropinosomes of all strains, except Nramp1-null mutant, suggesting that Nramp1 is essential for iron export. Mn or Cu did not affect de-quencing, suggesting that their transport is Nramp1-independent. In calcein-loaded Xenopus oocytes expressing Nramp1, Fe2+ or Mn2+, not Fe3+, led to fluorescence-quenching. Cu2+ favoured iron transport inside-out. In electrophysiological studies, Fe2+, Cd and Co, but not Fe3+ or Cu, elicited inward currents, confirming that Nramp1 does not transport Fe3+ and Cu. Radiochemical studies showed that Fe2+ transport is proton-dependent, and competed by Mn, Cd, Ni and Co, to a lower extent by Zn and Cu. CONCLUSION: Dictyostelium Nramp1 is an electrogenic proton-dependent iron transporter with cation selectivity comparable to mammalian DMT1. It transports Fe2+, not Fe3+ and is essential for Fe2+ export from macropinosomes.
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