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

线粒体ABC转运蛋白Atm1在细胞铁硫和铁代谢中的功能机制和结构分析

• 批准号: 271743333
• 负责人: Professor Dr. Roland Lill
• 金额: --
• 依托单位: Institut für Klinische Zytobiologie und Zytopathologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/271743333?language=en
• 关键词: Mechanistic; structural; analysis; function; mitochondrial

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.

线粒体在细胞铁硫（Fe/S）蛋白的生物发生中起着重要的作用。这些蛋白位于线粒体、细胞质和细胞核中，在呼吸、代谢、蛋白质翻译、DNA合成和DNA修复中起着重要作用。它们的Fe/S辅助因子通过复杂的机制组装并插入载脂蛋白中。线粒体铁硫簇（ISC）组装机制成熟了细胞器内铁/硫蛋白，但也是细胞质和核铁/硫蛋白组装不可或缺的。它合成一种称为X-S的含硫和谷胱甘肽因子，该因子由线粒体ISC输出装置输出到细胞质，在细胞质中，X-S被CIA（细胞质铁硫蛋白组装）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Atm1。其功能缺陷与细胞质核铁/硫蛋白和细胞铁调节缺陷有关。其人类同源物ABCB7的突变导致铁储存病x连锁铁母细胞性贫血和小脑性共济失调（XLSA/A）。我们最近解决了无核苷酸Atm1的晶体结构与不结合谷胱甘肽。尽管其在生理上具有重要意义，但ISC输出反应和Atm1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ISC输出反应和Atm1功能的机制阐明。根据我们最近的研究，我们想用分离的线粒体和细胞质[2Fe-2S]蛋白或早期作用的CIA成分在功能上重建输出过程，这些成分可能是X-S的受体。该系统将用于分离和表征X-S。我们进一步希望扩展胞质[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，对胞质铁/硫蛋白的这一分支尚不清楚。将进行进一步的结构和突变研究，以揭示Atm1的转运周期。研究的目的是精确定义底物结合袋，Atm1膜部分的出口通道，以及疾病相关突变的功能含义。结合底物和/或不同核苷酸的Atm1结构可以进一步识别其运输周期的快照。总体而言，该项目旨在提供ISC出口过程和Atm1作用的机械和结构视图。

项目成果

Structural and functional diversity calls for a new classification of ABC transporters.

• DOI: 10.1002/1873-3468.13935
• 发表时间: 2020-12
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Thomas C;Aller SG;Beis K;Carpenter EP;Chang G;Chen L;Dassa E;Dean M;Duong Van Hoa F;Ekiert D;Ford R;Gaudet R;Gong X;Holland IB;Huang Y;Kahne DK;Kato H;Koronakis V;Koth CM;Lee Y;Lewinson O;Lill R;Martinoia E;Murakami S;Pinkett HW;Poolman B;Rosenbaum D;Sarkadi B;Schmitt L;Schneider E;Shi Y;Shyng SL;Slotboom DJ;Tajkhorshid E;Tieleman DP;Ueda K;Váradi A;Wen PC;Yan N;Zhang P;Zheng H;Zimmer J;Tampé R
• 通讯作者: Tampé R

Biophysical methods toolbox to study ABC exporter structure and function

研究 ABC 输出蛋白结构和功能的生物物理方法工具箱

• DOI: 10.1515/hsz-2016-0244
• 发表时间: 2017
• 期刊: Biological Chemistry
• 影响因子: 3.7
• 作者: Marcellino T.;Srinivasan V.
• 通讯作者: Srinivasan V.