Askwith C C, Kaplan J
Division of Immunology and Cell Biology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
J Biol Chem. 1998 Aug 28;273(35):22415-9. doi: 10.1074/jbc.273.35.22415.
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.
酵母中的高亲和力铁转运由两种蛋白质Fet3p和Ftr1p介导。多铜氧化酶Fet3p被认为可将细胞外的亚铁离子转化为铁离子，然后铁离子通过通透酶Ftr1p穿过质膜。Fet3p能够氧化其他底物，如对苯二胺，并且铁转运所必需的是否是其氧化酶活性仍是一个问题。Ftr1p定位于细胞表面也需要Fet3p，这使得难以证明Fet3p氧化酶在高亲和力铁转运中的直接作用。为了产生特异性缺乏氧化酶活性的Fet3p，我们使用定点诱变来改变Fet3p中被认为赋予铁氧化酶活性的残基。这些取代导致对苯二胺和氧化酶活性的丧失或保留，表明Fet3p作为氧化酶的能力涉及其他氨基酸。然而，无活性的Fet3p确实介导了Ftr1p定位于细胞表面，但没有介导高亲和力铁转运。这些观察结果表明，Fet3p的氧化酶活性是高亲和力铁转运内在所需的。
