Abstract
The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.
Highlights
§ To whom correspondence should be addressed: Div. of Immunology and Cell Biology, Dept. of Pathology, University of Utah, College of Medicine, Salt Lake City, UT 84132
Evidence that suggested that Fet3 protein (Fet3p) is a ferroxidase included its homology to the multicopper oxidase family [6], a copper requirement for high affinity iron transport [6, 15], and the demonstration that a functioning Fet3p confers iron-dependent oxygen consumption in yeast [9]
The present study confirms the identity of Fet3p as a ferroxidase; Fet3p purified to apparent homogeneity contained copper and demonstrated ferroxidase activity in vitro
Summary
§ To whom correspondence should be addressed: Div. of Immunology and Cell Biology, Dept. of Pathology, University of Utah, College of Medicine, Salt Lake City, UT 84132. In an effort to further define the mechanism of high affinity iron transport, Fet3p has been purified to homogeneity, and its properties have been studied. Purified Fet3p is an oxidoreductase similar to other multicopper oxidases. We further demonstrate that in contrast to most multicopper oxidases, Fet3p has ferroxidase activity. Fet3p and ceruloplasmin (the only two multicopper oxidases known to be ferroxidases) may be functional homologues. The ability of ceruloplasmin to load iron onto apotransferrin is critical for its in vivo function. We demonstrate that the oxidation of ferrous ions by Fet3p can be coupled to the loading of apotransferrin. This observation suggests that a stable physical complex between the ferroxidase and the ferric acceptor may not be required for transfer of ferric iron
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