Mitochondria-cytoplasm interactions for cytosolic Fe-S cluster assembly

细胞质 Fe-S 簇组装的线粒体-细胞质相互作用

• 批准号: 10390734
• 负责人: ANDREW B. DANCIS
• 金额: $ 12.88万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10390734
• 关键词: Anabolism; Biogenesis; Biological Assay; Cell Line; Cell Survival; Cell physiology; Chemicals; Complex; Cytoplasm; Cytosol; Detection; Eukaryota; Genomic Instability; Human; Impairment; Inductively Coupled Plasma Mass Spectrometry; Inner mitochondrial membrane; Iron; Iron-Sulfur Proteins; Mammalian Cell; Mitochondria; Molecular Sieve Chromatography; Nature; Pathway interactions; Process; Production; Protein Biosynthesis; Proteins; Ribosomes; Role; Saccharomyces cerevisiae; Site; Sulfur; Testing; Transfer RNA; Work; Yeasts; cofactor; glutaredoxin; overexpression; scaffold; spectroscopic survey

Abstract In eukaryotes, the biosynthesis of essential Fe-S cluster cofactors is compartmentalized with a mitochondrial iron-sulfur cluster (ISC) machinery and a cytosolic iron-sulfur protein assembly (CIA) machinery. We found that isolated cytosol by itself cannot synthesize Fe-S clusters. Addition of mitochondria to the cytosol, however, allows this process to proceed. Similarly, thiolation of tRNAs is critical for accurate protein synthesis, and isolated cytosol alone cannot thiolate tRNAs but will do so upon addition of mitochondria. We found that the ISC machinery in mitochondria generates two distinct intermediates, Sint and (Fe-S)int. These intermediates are exported to the cytosol by the Atm1 transporter in the mitochondrial inner membrane. Once exported, Sint is utilized for tRNA thiolation and (Fe-S)int is utilized by the CIA machinery for cytosolic Fe-S cluster assembly. Aim 1 is to delineate the pathways for synthesis of Sint vs. (Fe-S)int. The site will be ascertained at which the pathway for Sint formation bifurcates from the ISC pathway for mitochondrial Fe-S cluster synthesis. The site at which (Fe-S)int formation bifurcates will likewise be determined. Mitochondria will be isolated from S. cerevisiae strains lacking components of the ISC machinery and they will be tested in mitochondria-cytosol mixing assays. Aim 2 is to demonstrate a direct role for Atm1 in exporting both Sint and (Fe-S)int. We will determine if the intermediates accumulate in Atm1-depleted mitochondria and if newly imported Atm1 into these mitochondria can restore the export process. Atm1 will be overexpressed to ascertain if this alters competition of Sint and (Fe- S)int for export. The exported and active (Fe-S)int, but not Sint, may contain bound GSH and this will be tested. Aim 3 is to define initial interactor(s) of (Fe-S)int in the cytosol. We will determine whether the glutaredoxins Grx3/4 and/or the Cfd1/Nbp35 scaffold complex interact with purified (Fe-S)int to build their own Fe-S clusters. Aim 4 is to purify and characterize Sint and (Fe-S)int exported from mitochondria. Exported intermediates will be purified by size exclusion chromatography, and active fractions will be characterized by ICP-MS, ESI-MS, and other spectroscopic studies with an aim to identifying the chemical composition of the intermediates. Aim 5 is to define whether mitochondria isolated from mammalian cell lines (CAD or HEK293) also produce and export two distinct intermediates, Sint and (Fe-S)int, and whether they can be swapped for the yeast equivalents in functional assays. Mitochondria lacking ABCB7 (human Atm1) will be tested for their ability/inability to generate Sint and (Fe-S)int intermediates and to export them for cytosolic use. The significance of this work derives from the critical nature of the processes that lie downstream of the exported intermediates. Impaired thiolation of cytosolic tRNAs (downstream of Sint) leads to disruption of protein synthesis. Impaired cytosolic Fe-S cluster assembly (downstream of (Fe-S)int) may create a cellular catastrophe due to genomic instability. Understanding the mechanistic details of mitochondrial production and export of Sint and (Fe-S)int along with detection/identification of these exported species as proposed here is highly significant.

抽象的 在真核生物中，必需的Fe-S簇辅因子的生物合成用线粒体划分了 铁硫簇（ISC）机械和胞质铁硫蛋白组件（CIA）机械。我们发现 分离的细胞质本身不能合成Fe-S簇。但是，在细胞质中添加线粒体允许 这个过程要进行。同样，TRNA的硫醇对准确的蛋白质合成至关重要，并且分离 仅胞质溶胶不能硫醇含量，但在添加线粒体后会这样做。我们发现ISC 线粒体中的机械产生了两个不同的中间体，SINT和（Fe-s）int。这些中间体是 通过ATM1转运蛋白在线粒体内膜中导出到细胞质。一旦出口，SINT是 CIA机械用于胞质Fe-S簇组装，用于tRNA硫醇化和（Fe-S）INT。 目的1是描述合成SINT与（Fe-S）Int的途径。将确定该网站 SINT形成的途径是从ISC途径分叉的线粒体Fe-S簇合成的途径。网站位于 同样将确定哪个（Fe-S）int形成分叉。线粒体将从酿酒酵母中分离出来 缺少ISC机械组件的菌株，将在线粒体 - 胞质混合测定中进行测试。 AIM 2是展示ATM1在导出SINT和（Fe-S）Int中的直接作用。我们将确定是否 中间体积累在耗尽的线粒体中，如果新进口的ATM1进入这些线粒体 可以恢复出口过程。 ATM1将过表达以确定这种SINT和（fe- S）INT出口。导出和活性（Fe-S）INT（而不是SINT）可能包含绑定的GSH，这将进行测试。 AIM 3是定义细胞质中（Fe-S）INT的初始相互作用。我们将确定是否谷物毒素 GRX3/4和/或CFD1/NBP35脚手架复合物与纯化（Fe-S）INT相互作用，以构建自己的Fe-S簇。 目的4是纯化和表征SINT和（Fe-s）Int从线粒体导出的。出口中间体会 通过尺寸排除色谱法纯化，主动部分将以ICP-MS，ESI-MS为特征 和其他光谱研究，目的是识别中间体的化学组成。 AIM 5是定义从哺乳动物细胞系分离的线粒体（CAD或HEK293）是否也会产生 并导出两个不同的中间体，SINT和（Fe-S）INT，以及是否可以换成酵母 功能测定中的等效物。缺乏ABCB7的线粒体（人ATM1）将进行测试 能力/无法生成SINT和（Fe-S）INT中间体并将其导出以供胞质使用。 这项工作的重要性来自于下游过程的批判性质 导出的中间体。胞质TRNA的硫醇化受损（SINT的下游）导致蛋白质破坏 合成。胞质Fe-S簇组装受损（（Fe-S）INT的下游）可能会造成细胞灾难 由于基因组不稳定。了解线粒体生产和SINT出口的机械细节 （Fe-S）INT以及此处提出的这些出口物种的检测/识别非常重要。