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Supp. Figure S1. The MFS fold. Ribbon representation of two of the three known 3D structures from MFS proteins, after superimposition. The two structures are shown viewed from the membrane plane (top) and from the extracellular side (bottom). The MFS fold is organized around to equivalent domains, each possessing six helices (helices H1 to H6 and H7 to H12), organized as two three-helix bundles. Ribbons are rainbow colored (from blue to red) in the two halves, in order to highlight the duplication and the equivalent helices. (A) 3D structure of the glycerol-3-phosphate transporter (GlpT) from E. coli in an inward-facing configuration (pdb 1pw4). (B) 3D structure of the multidrug transporter EmrD from E. coli in an intermediate configuration, thought to represent an occluded state (pdb 2gfp). The two following segments from H2 (1pw4: 78-93, 2gfp: 58-73) and H8 (1pw4: 303-312, 2gfp 253-262) were used for the superimposition, which led to an overall good match. The 3D structure of E. coli GlpT (pdb 1pw4) is very similar (1.8 Å RMSD) to that of sugar free lactose permeaseLacY from E. coli (pdb 2cfq).

Supp. Figure S2 (A-B). Sequence alignment of human ferroportin with E. Coli EmrD. Representative members of both the EmrD and ferroportin (SLC40A1_HUMAN) families were chosen to illustrate the sequence conservation in each family and how the sequence profiles match together. The observed secondary structures of E. coli EmrD (pdb 2gfp) are shown above its sequence (H stands for Helix, ES for Extra-cellular segment and IS for Intra-cellular segment). Below the set of ferroportin sequences are reported with green squares amino acids that participate in the pore (also see Fig. 3). Amino acids associated with disease-causing mutations are boxed in red. Tyrosines 302 and 303 and lysines between positions 229 and 269 are in blue. Amino acids chosen for experimental investigation are shown in purple. Shaded boxes indicate the positions of loops that could not be modeled and were thus omitted from the model.

Supp. Figure S3. Topology of the human ferroportin. Liu et al. investigated topology of the mouse ferroportin using epitope-tagged proteins, and cell surface biotinylation and labeling of native or introduced cysteines [Liu et al., 2005]. Human (571 AA, UniProt accession number Q9NP59) and mouse ferroportin (570 AA, UniProt accession number Q9JHI9) share 90% of sequence identity and most of the changes are conservative and moreover mostly concentrated in N- and C-terminal segments. The positions of the epitope tag inserts (HA or myc) used by Liu et al. to confirm sequences of the intracellular (IS) and extracellular segments (ES) of ferroportin are indicated by blue arrows. The location for each epitope insertion is done in parentheses. ER means that the tagged proteins did not reach the cell surface, but were retained within the Endothelial Reticulum. The positions of the native cysteines mutated by Liu et al. are indicated in red (C326: p.Cys326) or in orange (C205: p.Cys205), whereas the positions of the cysteines introduced into several predicted interdomains regions are indicated in pink. The figure also represents positions of key amino acids for the down-regulation of ferroportin by hepcidin (in red), and positions of certain amino-acids known to be mutated in ferroportin disease (loss-of-function mutations, in black) or hemochromatosis patients (gain-of-function mutation, in brown). The main differences between the two proposed models of ferroportin topology are underlined. Note that human ferroportin is composed of 571 aa, while mouse ferroportin is composed of 571 aa; the difference has been taken into account in the two models. . Amino acids are presented using the IUPAC single letter codes (C: Cys, D: Asp, G: Gly, H: His, K: Lys, V: Val, Y: Tyr).

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