Hepcidin interaction with ferroportin in the Xenopus oocyte expression system

Abstract

The cellular iron‐export protein ferroportin (Fpn), expressed in enterocytes and macrophages, is essential for iron homeostasis. Fpn operates under the control of the iron‐regulatory hormone hepcidin. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transport protein [Drakesmith H et al (2015) Cell Metab 22, 777–787]. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin treatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux that was not dependent on endocytosis, a conclusion based on the following four observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10 = 2.5 ± [SEM] 1.4 over the range 17–27 °C) whereas endocytosis is expected to be substantially more temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–2617]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the K8R mutant, in which 8 Lys residues required for ubiquination and endocytosis of Fpn were mutated to Arg, to the same degree as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and K8R‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after 30‐min treatment with hepcidin. Our data reveal that hepcidin can directly block Fpn activity independent of Fpn internalization. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.Support or Funding InformationNIH–NIDDK grant R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.