Mechanistic and structural analysis of the function of the mitochondrial ABC transporter Atm1 in cellular iron-sulfur and iron metabolism

线粒体在细胞铁-硫（Fe/S）蛋白的生物合成中发挥重要作用。这些蛋白质位于线粒体、细胞质和细胞核中，并在呼吸、代谢、蛋白质翻译、DNA合成和DNA修复中完成重要任务。它们的Fe/S辅因子通过复杂的机制组装并插入脱辅基蛋白中。线粒体铁硫簇（ISC）组装机制成熟的细胞器内Fe/S蛋白，但也是必不可少的胞质和核Fe/S蛋白组装。它合成一种含硫和谷胱甘肽的因子，称为X-S，由线粒体ISC输出装置输出到胞质溶胶，在胞质溶胶中，X-S被CIA（胞质溶胶铁硫蛋白组装）机制的早期作用组分用于线粒体外Fe/S蛋白的成熟。ISC输出系统的核心组成部分是线粒体ABC转运蛋白Atm 1。其功能缺陷与胞质-核Fe/S蛋白和细胞铁调节的缺陷有关。其人类直系同源物ABCB 7的突变导致铁储存疾病X连锁铁粒幼细胞性贫血和小脑共济失调（XLSA/A）。我们最近解决了无核苷酸Atm 1的晶体结构与结合谷胱甘肽。尽管其最重要的生理意义，ISC输出反应和Atm 1功能的精确分子方面仍然知之甚少。因此，当前提案的中心目标是阐明ISC输出反应和Atm 1功能的机制。我们希望用分离的线粒体和胞质[2Fe-2S]蛋白或根据我们最近的研究可能作为X-S受体的早期作用CIA组分在功能上重建输出过程。该系统将用于分离和表征X-S。我们还希望扩大胞质[2Fe-2S]蛋白如何成熟的知识，以更好地表征CIA机制的早期部分。迄今为止，对胞质Fe/S蛋白生物发生的这个分支还一无所知。将进行进一步的结构和突变研究，以解开Atm 1的运输周期。调查的目的是在精确定义的底物结合口袋，出口通道内的膜部分的Atm 1，和疾病相关的突变的功能意义。Atm 1结构与绑定基板和/或不同的核苷酸可以识别其运输周期的进一步快照。总体而言，该项目旨在提供ISC导出过程和Atm 1作用的机械和结构视图。

Mitochondria perform an essential function in the biogenesis of cellular iron-sulfur (Fe/S) proteins. These proteins are located in mitochondria, cytosol and nucleus, and fulfill important tasks in respiration, metabolism, protein translation, DNA synthesis and DNA repair. Their Fe/S co-factors are assembled and inserted into apoproteins by complex machinery. The mitochondrial iron-sulfur cluster (ISC) assembly machinery matures intra-organellar Fe/S proteins, yet is also indispensable for cytosolic and nuclear Fe/S protein assembly. It synthesizes a sulfur- and glutathione-containing factor termed X-S that is exported by the mitochondrial ISC export apparatus to the cytosol where X-S is used by early acting components of the CIA (cytosolic iron-sulfur protein assembly) machinery for maturation of extra-mitochondrial Fe/S proteins. The central component of the ISC export system is the mitochondrial ABC transporter Atm1. Its functional deficiency is associated with defects in cytosolic-nuclear Fe/S proteins and in cellular iron regulation. Mutations in its human ortholog ABCB7 cause the iron-storage disease X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). We recently solved the crystal structure of nucleotide-free Atm1 with and without bound glutathione. Despite its utmost physiological importance, precise molecular aspects of the ISC export reaction and Atm1 function are still poorly understood. Hence, the central goal of the current proposal is the mechanistic elucidation of the ISC export reaction and of Atm1 function. We want to functionally reconstitute the export process with isolated mitochondria and cytosolic [2Fe-2S] proteins or early-acting CIA components that according to our recent studies may function as acceptors of X-S. This system will be used to isolate and characterize X-S. We further want to expand the knowledge of how cytosolic [2Fe-2S] proteins are matured to better characterize the early part of the CIA machinery. Nothing is known so far about this branch of cytosolic Fe/S protein biogenesis. Further structural and mutational studies will be performed to unravel the transport cycle of Atm1. The investigations aim at the precise definition of the substrate binding pocket, the export channel within the membrane part of Atm1, and of the functional implication of disease-relevant mutations. Atm1 structures with bound substrate and/or different nucleotides may identify further snapshots of its transport cycle. Overall, the project aims to provide a mechanistic and structural view of the ISC export process and the role of Atm1.