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 (Fpn). To study hepcidin-Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin-induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C-terminus. Once the NanoLuc-tagged Fpn 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 NanoLuc-tagged Fpn and an enhanced green fluorescent protein (EGFP)-tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin-induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin-Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc-tagged wild-type Fpn and an EGFP-tagged hepcidin-insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin-induced internalization of wild-type Fpn, suggesting that wild-type Fpn and mutant Fpn are functionally independent.