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Studies on the roles of human ferroportin and hephaestin in iron homeostasis

University of British Columbia

Ferroportin-1 (Fpn1) is a highly conserved 571-amino acid protein with the human, mouse, and rat polypeptides having 90–95% sequence identity at the amino acid level. Disruption of Fpn1 expression in zebrafish and mice results in embryonic lethality, while conditional knockout of the gene encoding Fpn1 in the intestine at the postnatal stage leads to severe iron deficiency anemia and iron accumulation in duodenal enterocytes. These studies suggest that Fpn1 is the major, if not the sole, exporter protein for iron. Export of dietary iron is thought to be facilitated by hephaestin (Hpn), a multicopper ferroxidase in the basolateral membrane of duodenal enterocytes. However, the precise mechanism of iron transport by Fpn1 and Hpn remains to be elucidated. The absence of an iron export mechanism in Saccharomyces cerevisiae was utilized to explore how Fpn1 and Hpn function in iron export. Unlike humans, S. cerevisiae transports excess iron into a storage vacuole through the vacuolar iron transporter, Ccc1p. The Δccc1 mutant fails to store excess iron; as a result, iron accumulates in the cytosol and cells die due to oxidative stress when exposed to high concentrations of extracellular iron. By expressing recombinant human Fpn1 and Hpn in S. cerevisiae, an iron export system was introduced. The functionality of rhHpn in S. cerevisiae was confirmed by both a ferrozine ferroxidase assay and a transferrin iron-loading assay. The effects of the individual or both recombinant proteins on iron sensitivity of the Δccc1 yeast with an additional fet3 (the yeast homolog of Hpn) deletion were evaluated. Recombinant human Fpn1 and Hpn suppressed the lethal phenotype of the Δccc1 mutant while co-expression of rhHpn with rhFpn1 led to a stronger rescue phenotype of the Δccc1 mutant under concentrations of high extracellular iron. The expression of rhFpn1 and / or rhHpn also relieved the copper sensitive Δfet3 mutant from copper stress. A physical interaction between rhFpn1 and rhHpn was demonstrated by cross-linking with BS³. These studies advance our knowledge of the roles Fpn1 and Hpn play in human enterocytes and suggest that these proteins are intimately involved in iron export from enterocytes into the blood.

Text

2010-10-14

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/29161

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/