A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin

Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin ( F pn). To study hepcidin– F pn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin‐induced F pn internalization by fusing a small nanoluciferase ( N ano L uc, 171 amino acids) at the F pn C ‐terminus. Once the N ano L uc‐tagged F pn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a N ano L uc‐tagged F pn and an enhanced green fluorescent protein ( EGFP )‐tagged F pn by the use of an inducible bidirectional promoter, we could measure the hepcidin‐induced F pn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged N ano L uc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin– F pn interaction qualitatively and quantitatively. Through coexpression of a N ano L uc‐tagged wild‐type F pn and an EGFP ‐tagged hepcidin‐insensitive mutant [C326S] F pn, we demonstrated that the mutant Fpn had no effect on hepcidin‐induced internalization of wild‐type F pn, suggesting that wild‐type F pn and mutant F pn are functionally independent.