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	Fig. 1. Calcein is taken up by macropinocytosis and colocalizes with TRITC–dextran in vesicles decorated with Nramp1. (A) Exponentially growing wild-type (Ax2) or Nramp1-KO cells were washed, resuspended in low fluorescence medium and plated on glass coverslips equipped with a Plexiglas ring. Upon addition of calcein, consecutive confocal images of single cells were taken at the indicated times (in seconds). Calcein fluorescence and corresponding phase-contrast images are shown. Arrowheads point to macropinocytic cups and vesicles. (B) AX2 wild-type cells were incubated with calcein and TRITC–dextran under shaking for 30†min, then washed and resuspended in Soerensen phosphate buffer. After plating on glass coverslips, confocal series images were taken. Coincidence of calcein and TRITC–dextran was detected in almost all vesicles, reflected in the yellow fluorescence in the merge with the phase-contrast image, indicating that calcein is taken up by macropinocytosis. (C) AX2 cells expressing Nramp1– or Nramp2–RFP were incubated with calcein under shaking for 30†min, washed and resuspended in Soerensen phosphate buffer. After plating on glass coverslips, confocal images were taken to detect calcein and the RFP fluorescence of the proteins. The merge with phase-contrast image is also shown. Calcein is found in vesicles decorated with Nramp1. In cells expressing Nramp2–RFP, calcein is not found at the bottom of the cell, where the contractile vacuole is located. In upper stacks (i.e. towards the top of the cell), calcein vesicles are evident, but they are not decorated with Nramp2, as expected given that Nramp2 is not recruited to macropinosomes. Numbers indicate the distance in µm from the bottom. Scale bars: 5 µm.
	Fig. 2. Iron-chelated calcein is de-quenched in macropinosomes of wild-type or Nramp2-KO, but not Nramp1-KO, cells. (A) Quantification of calcein fluorescence quenching by divalent metals. Calcein dissolved in NaOH was diluted (25†µM) in 0.1†M acetate buffer, pH 5.0 or 0.05†M PIPES or HEPES, at pH 6.0 or 7.0, respectively. The different buffers were required to maintain the right pH. Calcein was then titrated with increasing concentrations of FeCl3, FeCl2, CuCl2 or MnCl2. The fluorescence intensity (λex=485†nm, λem=535†nm) was measured in non-treated white multiwell plastic plates (NUNC) in a Perkin-Elmer EnSpire Multimode Plate Reader, and the quenching expressed as a percentage of fluorescence intensity in controls containing the same amount of calcein in the absence of any divalent metal (n=1 experiment in duplicate). (B) Exponentially growing cells were plated on a glass coverslip and incubated with a solution of calcein (CAL) and saturating amount of Fe(Cl)2, which completely quenches calcein fluorescence (note the black surrounding the cells at time 0). As time progresses (shown in minutes), spots of fluorescent calcein in AX2 cells appear and increase in number and intensity, indicating iron detachment from calcein and its efflux in the cytosol. This does not occur in the Nramp1-KO mutant. (C) AX2, Nramp1-KO and Nramp2-KO cells were treated as in B but under shaking. At the times indicated, the cells were washed and their fluorescence quantified in a flow cytometer. Calcein fluorescence mean value increases steadily in AX2 and Nramp2-KO cells, but not in Nramp1-KO cells. (D) Cells were treated as in C in the presence of the indicated divalent metals, which all quench calcein fluorescence. After 30†min of incubation, cells were washed and the mean fluorescence value determined by flow cytometry. Calcein de-quenching (fluorescence recovery) was expressed relative to maximal fluorescence (i.e. to the value of calcein fluorescence in each cell type without added metal). Calcein de-quenching occurs also in Nramp1-KO cells incubated with manganese and copper, but not iron, suggesting that other transporters might be active for these metals. In the case of manganese, acidification might be sufficient to induce de-quenching (see Fig. 1). In C and D, mean±s.e.m. values of at least three independent experiments are shown. *P<0.05 or **P<0.01 (two-tailed Student's t-test, assuming unequal variance). Scale bars: 5 μm.
	Fig. 3. Live imaging of divalent metal transport in Xenopus oocytes using calcein fluorescence. Xenopus oocytes expressing c-Nramp1, c-Nramp2 or rDMT1 were injected with calcein and perfused with a solution containing or not the indicated metal. Confocal series images were taken at the time indicated. Left, in the absence of any metal, calcein fluorescence was stable in all oocytes for all the length of the experiment. In the presence of Mn(Cl)2 or Fe(Cl)2 fluorescence decreased within minutes, although this was less evident for c-Nramp2-expressing oocytes. Right, the fluorescence changes over time were quantified by measuring the ratio of fluorescence intensity at the time indicated (F) versus fluorescence at time 0 (F0). Mean±s.d. are shown for each group of oocytes [c-Nramp1 n values are: Mn, 3; Fe(II), 8; Fe(III), 4; Cu, 5; c-Nramp2 n values are: Mn, 2; Fe(II), 7; Fe(III), 11; Cu, 5; rDMT1 n values are: Mn, 2; Fe(II), 8; Fe(III), 8; Cu, 7]. Statistical analysis was undertaken at the 9-min time point using a two-tailed Student's t-test, assuming unequal variance: Fe(II) versus control was P<0.05 in all cases; Cu versus control was P<0.05 for c-Nramp1. Scale bars: 0.5 mm.
	Fig. 4. Fe and Mn elicit pH-dependent transport currents in oocytes expressing c-Nramp1 or rDMT1. (A,B) Oocytes expressing c-Nramp1 or rDMT1 were clamped at a constant voltage of −40†mV in sodium solution at (A) pH 6.0 or (B) at different pH, and perfused with the indicated metal. Currents were recorded as described in the Materials and Methods. Effects of increasing metal ion concentrations and pH on transport currents for a single oocyte are shown on top and the mean±s.e.m. values for 8 to 20 oocytes for each condition in three different experiments are shown at the bottom of the figure. P<0.05 for c-Nramp1 and rDMT1 compared to control oocytes or at pH 5.5 versus pH 7.6. Both Mn and Fe evoked currents mediated by c-Nramp1 and rDMT1, which are maximal at pH 5.5, smaller at higher pH and switch outwardly at basic pH, at least for c-Nramp1. (C) Oocytes were perfused with Mn(Cl)2 in sodium or TMA solution. The outward current evident with c-Nramp1 disappears if Na is replaced by TMA, suggesting the presence of a Na leakage current that is blocked by the presence of the co-transported ion (Mn). For all the traces recorded, the holding current (Ih) is indicated. Mean±s.e.m. values of 5 to 10 experiments with 10 to 50 oocytes are shown. P<0.05 for c-Nramp1 and DMT1 compared to control oocytes or at pH 7.6.
	Fig. 5. Differential effects on transport currents by metal ions in Xenopus oocytes expressing c-Nramp1 or rDMT1. Oocytes expressing c-Nramp1 or rDMT1 were clamped at a constant voltage of −40†mV and perfused with the indicated metal at a concentration of 0.1†mM in either sodium or TMA solution at pH 6.0. Currents were recorded as described in the Materials and Methods. Top, mean±s.e.m. values of a variable number of experiments for each condition, with four oocytes in three experiments and up to 50 oocytes in 10 experiments, and, bottom, recording of single oocytes in TMA. For all the traces recorded, the holding current (Ih) was reported.
	Fig. 6. Radiochemical uptake assays with Xenopus oocytes expressing c-Nramp1, c-Nramp2 or rDMT1. (A) Control oocytes (non inj.) or oocytes injected with cRNA for c-Nramp1 or rDMT1 (cRNA inj.) were incubated with 0.1†mM 55Fe(Cl)2 in the presence or not of 1†mM Mn(Cl)2 as described in the Materials and Methods. At the end of incubation, the oocytes were washed and dissolved in SDS, and the accumulated radioactivity was counted in a liquid scintillation counter. Mean±s.e.m. values for groups of 8–10 oocytes, in a representative experiment of three independent, are shown. Both c-Nramp1 and rDMT1 mediate iron uptake, which is inhibited by Mn. P<0.001 for Nramp1 control versus non inj. Control; P<0.003 for Nramp1 Mn versus Nramp1 control (Student's t-test). (B,C) Oocytes injected or not with c-Nramp1 were assayed for transport of 55Fe(Cl)2 (B) at different pH or (C) in the presence of different metal ions. In B, mean±s.e.m. values for groups of 8–10 oocytes in a representative experiment of three independent are shown. P<0.05 for cRNA inj. pH 5.5 versus cRNA inj. pH 6.5; P<0.004 for cRNA inj. pH 5.5 versus cRNA inj. pH 8 (Student's t-test). In C, data represent 55Fe uptake in the absence (control) or presence of the indicated divalent metal at a concentration of 1†mM. Data are the mean±s.e.m. for groups of 8–10 oocytes in three separate experiments. P<0.04 for each metal versus control (Student's t-test). Uptake by c-Nramp1 is pH dependent, being maximal at pH 5.5, and inhibited by all metals tested, although to a lower extent for zinc. (D) Oocytes injected with c-Nramp2 were tested for transport of 0.1†mM 55Fe(Cl)2 at the pH values indicated. Mean±s.e.m. values for groups of 8–10 oocytes in a representative experiment of three independent are shown. P<0.001 for cRNA inj. pH 5.5 versus cRNA inj. pH 7.5; c-Nramp2 induces significant iron accumulation only at pH 7.5 (Student's t-test).
	Fig. 7. Sequence alignment. Sequences of Dictyostelium Nramp1 and Nramp2, human Nramp1 and DMT1 (formerly Nramp2) (isoform1), rat DMT1 and Staphylococcus capitis DMT were aligned with ClustalW. Identical or homologous residues are shown in sky blue or light blue, respectively, and non-conserved residues are in white. Only the first ten transmembrane domains are shown, with the numberings reporting the amino acid residues of each protein. The predicted transmembrane domains are as described previously (Czachorowski et al., 2009; Ehrnstorfer et al., 2014). The stars point to the four amino acid residues coordinating iron binding, and the triangles to the two histidine residues required for proton dependency of transport (Czachorowski et al., 2009; Ehrnstorfer et al., 2014). Amino acids whose spontaneous or induced mutations in human or rodent DMT1 result in reduction of uptake and/or changes in specificity or pH dependency are indicated by circles (see: Fleming et al., 1997; Lam-Yuk-Tseung et al., 2003; Cohen et al., 2003; Courville et al., 2006; Beaumont et al., 2006; Czachorowski et al., 2009; Iolascon et al., 2006).

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