Site-directed mutagenesis of the yeast multicopper oxidase Fet3p.
High affinity iron transport in yeast is mediated by two proteins, Fet3p and Ftr1p. The multicopper oxidase Fet3p is thought to convert extracellular ferrous iron to ferric iron, which then crosses the plasma membrane through the permease Ftr1p. Fet3p is capable of oxidizing other substrates, such as p-phenylenediamine, and there is still a question of whether it is the ferroxidase activity that is essential for iron transport. Fet3p is also required for Ftr1p localization to the cell surface, making it difficult to prove a direct role for Fet3p oxidase in high affinity iron transport. In an attempt to generate Fet3p specifically lacking ferroxidase activity, we used site-directed mutagenesis to alter residues within Fet3p that had been suggested to impart iron oxidase activity. These substitutions resulted in either a loss or retention of both p-phenylenediamine and ferroxidase activities, indicating that the ability of Fet3p to act as a ferroxidase involves other amino acids. Inactive Fet3p, however, did mediate Ftr1p localization to the cell surface but did not mediate high affinity iron transport. These observations indicate that the ferroxidase activity of Fet3p is intrinsically required for high affinity iron transport.
Division of Immunology and Cell Biology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
C C
CC
Askwith
J
J
Kaplan
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002570:Ceruloplasmin; D017931:DNA Primers; D007501:Iron / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D007501:Iron; D010088:Oxidoreductases; C024748:copper oxidase; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae