Fet3 (Ferroxidase) and Ftrl (Permease) in Iron Uptake

描述（由申请人提供）：真核铁代谢包括两个过程：氧化还原循环和运输。“游离”铁在真核生物血浆和一些细胞膜上的运输是这种代谢的一个范例。因此，环境铁离子的吸收首先涉及质膜铁还原酶对其的还原。生成的Fe2+可以作为多铜氧化酶的底物——一种铁氧化酶，它将O2的还原与4Fe3+的生成结合起来。然后，这个铁是铁渗透酶的配体，铁渗透酶通过质膜运输铁。酵母（Saccharomyces cerevisiae）的高亲和力铁摄取表现出所有这些特征。金属还原酶Fre1p产生Fe2+，该Fe2+是铜蓝蛋白同源物Fet3p氧化铁的底物，并通过Ftr1p促进渗透。在酵母中，和在肠上皮中一样，铁氧化和渗透步骤在严格的代谢意义上是耦合的：渗透需要铁氧化。这种耦合提出了本研究的一个主要假设：在Fet3p， Ftr1p体系中，fe3p铁氧化酶反应的三铁产物被输送到Ftr1p，以进行随后的跨膜运输。这个模型的模板是铁进出铁蛋白（Ft）核心的运动。这一假设要求，除了铁氧化和渗透本身所需的结构基序外，Fet3p和Ftr1p都具有参与这一通道过程的氨基酸残基。也可能存在与这两个过程耦合相关的基序。本研究的目的是利用生化、生物物理、遗传和细胞生物学方法对Fet3p、Ftr1系统进行全面、详细的结构功能分析。这些研究包括：野生型和突变型Fet3蛋白的动力学、光谱和晶体学研究；Ftr1p输铁突变体铁摄取动力学分析Fet3p和Ftr1p之间物理和功能相互作用的生化、遗传和荧光分析；以及铁氧化与摄取耦合的动力学和电生理分析。Fet3p， Ftr1p系统的结构-功能表征将为真核铁运输提供重要的新认识。

DESCRIPTION (provided by applicant): Eukaryotic iron metabolism involves two processes: redox cycling and trafficking. The transport of 'free' iron across eukaryotic plasma and some intracellular membranes is a paradigm of this metabolism. Thus, uptake of environmental Fe3+ involves first its reduction by a plasma membrane ferrireductase. The Fe2+ produced can be substrate for a multicopper oxidase - a ferroxidase - that couples the reduction of O2 to the production of 4Fe3+. This ferric iron is then ligand for an iron permease that transports the iron across the plasma membrane. High affinity iron uptake in the yeast, Saccharomyces cerevisiae, exhibits all of these features. The metalloreductase, Fre1p, produces the Fe2+ that is substrate for ferroxidation by Fet3p, a ceruloplasmin ortholog, with permeation facilitated by Ftr1p. In yeast, as in the intestinal epithelium, the ferroxidation and permeation steps are coupled in the strict metabolic sense: permeation requires ferroxidation. This coupling suggests a primary hypothesis of this research: in the Fet3p, Ftr1p system the ferric iron product of the Fet3p ferroxidase reaction is channeled to Ftr1p for subsequent transmembrane trafficking. A template for this model is the movement of iron into and out of the ferritin (Ft) core. This hypothesis requires that both Fet3p and Ftr1p possess amino acid residues that participate in this channeling process, in addition to those structural motifs required for ferroxidation and permeation per se. There also may be motifs associated with the coupling of these two processes. The objective of this research is a full and detailed structure-function analysis of the Fet3p, Ftr1 system using biochemical, biophysical, genetic and cell biology approaches. These include: kinetic, spectral and crystallographic studies of wild type and mutant Fet3 proteins; iron uptake kinetic analysis of Ftr1p iron trafficking mutants; biochemical, genetic and fluorescence analysis of the physical and functional interaction between Fet3p and Ftr1p; and kinetic and electrophysiologic analysis of the coupling of ferroxidation and uptake. This structure-function characterization of the Fet3p, Ftr1p system will provide significant new understanding of eukaryotic iron trafficking.