Cholesterol modulates the human FPN1 iron export function in plasma membrane liquid-ordered microdomains

Ferroportin 1 (FPN1) is the only known mammalian iron efflux transporter. This multi-pass membrane protein, which adopts the Major Facilitator Superfamily fold, is tightly controlled by serum hepcidin to assure maintenance of adequate cellular and systemic iron levels. Earlier studies have shown that cholesterol-lowering drugs can reduce FPN1 expression in liquid-ordered plasma membrane microdomains and its sensitivity to hepcidin. However, the molecular mechanism by which cholesterol depletion regulates the localization of FPN1 at the cell surface remains unknown. In biochemical experiments, we show that cholesterol depletion reduces the iron export function of FPN1. Repletion with cholesterol restores FPN1 activity. This is not observed with the diastereoisomer epicholesterol, suggesting a direct interaction between cholesterol and FPN1. Consistent with this, we demonstrate that mutants affecting the key tyrosine residues of three cholesterol-recognition amino acid consensus (CRAC/CARC) motifs have a negative impact on FPN1 activity, in manner that also decreases its abundance in ordered plasma membrane microdomains. A complementary structural analysis allows us to focus on a conserved CARC motif (CARC-1) located in a deep hydrophobic groove between transmembrane helices 1 and 5. Molecular docking suggests that this groove is well suited to cholesterol binding. All these findings indicate that the interaction between FPN1 and cholesterol is of major importance for the localization of FPN1 in ordered microdomains of the plasma membrane, which is necessary for its optimal activity, and so its responsiveness to hepcidin.