Site-directed mutagenesis of the yeast multicopper oxidase Fet3p
Site-directed mutagenesis of the yeast multicopper oxidase Fet3p
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DOI：
10.1074/jbc.273.35.22415
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发表时间：
1998-08-28
影响因子：
4.8
通讯作者：
Kaplan, J
中科院分区：
文献类型：
--
作者：
Askwith, CC;Kaplan, J
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Fet 3 p被认为将细胞外的二价铁转化为三价铁，然后通过渗透酶Ftr1 p穿过质膜，Fet 3 p能够氧化其他底物，例如对苯二胺，并且仍然存在一个问题是否是铁氧化酶活性对于铁转运至关重要。Fet3p也需要Ftr1p定位到细胞表面，使得难以证明Fet3p氧化酶在高亲和力铁转运中的直接作用。在试图产生特异性缺乏铁氧化酶活性的Fet3p中，我们使用定点诱变来改变Fet3p中已被建议赋予铁氧化酶活性的残基。这些取代导致对苯二胺和铁氧化酶活性的损失或保留，表明Fet3p作为铁氧化酶的能力涉及其他氨基酸。然而，非活性Fet3p介导Ftr1p定位到细胞表面，但不介导高亲和力铁转运。这些观察结果表明，铁氧化酶活性的Fet3p本质上是需要高亲和力铁运输。
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.