Purification and characterization of Fet3 protein, a yeast homologue of ceruloplasmin.
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.
de Silva
D
D
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Davis-Kaplan
S
S
Fergestad
J
J
Kaplan
J
J
D002570:Ceruloplasmin / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D010088:Oxidoreductases; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae