Functional studies of hephaestin in yeast: evidence for multicopper oxidase activity in the endocytic pathway

• Published in: Biochemical journal Vol. 375; no. Pt 3; pp. 793 - 798
• Main Authors: Li, Liangtao, Vulpe, Chris D, Kaplan, Jerry
• Format: Journal Article
• Language: English
• Published: England 01-11-2003
• Subjects: Animals; Biological Transport; Blotting, Western; Cell Division - drug effects; Ceruloplasmin - genetics; Ceruloplasmin - metabolism; Endocytosis; ferroxidase; Fet3 protein; Genetic Complementation Test; hephaestin; Iron - metabolism; Iron - pharmacology; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; multicopper oxidase; Mutation; Organelles - metabolism; Plasmids - genetics; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Transformation, Genetic
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20030866

Hephaestin is a mammalian gene that encodes a predicted multicopper oxidase required for intestinal iron export. To examine if hephaestin can act as a ferroxidase, we studied yeast strains transformed with plasmids containing both a full-length hephaestin and a hephaestin lacking a transmembrane domain. Yeast with a deletion in FET3, which encodes a cell-surface multicopper oxidase, cannot grow on low-iron media. Expression of full-length hephaestin could complement the low-iron growth phenotype of a Delta fet3 strain. Complementation of Delta fet3 cells by hephaestin required genes that encode proteins necessary for the copper loading of Fet3p, including CCC2 and GEF1. Expression of hephaestin in Delta fet3 cells led to an increase in both iron transport and oxidase activity. These results demonstrate that hephaestin is a copper-dependent protein. In contrast with Fet3p, which is found on the cell surface, hephaestin was co-localized with Pep12p-containing vesicles. Inhibition of endocytosis or deletion of both the vacuolar iron transporters ( SMF3 and FET5 / FTH1 ) prevented hephaestin from complementing the low-iron growth phenotype of Delta fet3 cells, suggesting that hephaestin is functioning within the endocytic apparatus.