SITE-DIRECTED MUTAGENESIS OF THE YEAST MULTICOPPER OXIDASE FET3P

High affinity iron transport in yeast is mediated by two proteins, Fet 3p and Ftr1p. The multicopper oxidase Fet3p is thought to convert extr acellular ferrous iron to ferric iron, which then crosses the plasma m embrane through the permease Ftr1p, Fet3p is capable of oxidizing othe r substrates, such as p-phenylenediamine, and there is still a questio n 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 speci fically lacking ferroxidase activity, we used site-directed mutagenesi s to alter residues within Fet3p that had been suggested to impart iro n oxidase activity. These substitutions resulted in either a loss or r etention of both p-phenylenediamine and ferroxidase activities, indica ting that the ability of Fet3p to act as a ferroxidase involves other amino acids. Inactive Fet3p, however, did mediate Ftr1p localization t o the cell surface but did not mediate high affinity iron transport. T hese observations indicate that the ferroxidase activity of Fet3p is i ntrinsically required for high affinity iron transport.