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 簇辅助因子的生物合成由线粒体划分 铁硫簇（ISC）机制和胞质铁硫蛋白组装（CIA）机制。我们发现 分离的细胞质本身不能合成Fe-S簇。然而，将线粒体添加到细胞质中可以 这个过程继续进行。同样，tRNA 的硫醇化对于准确的蛋白质合成至关重要，并且分离的 单独的胞质溶胶不能硫醇化 tRNA，但在添加线粒体后会硫醇化 tRNA。我们发现 ISC 线粒体中的机制产生两种不同的中间体：Sint 和 (Fe-S)int。这些中间体是 通过线粒体内膜中的 Atm1 转运蛋白输出至细胞质。一旦导出，Sint 用于 tRNA 硫醇化，(Fe-S)int 被 CIA 机器用于胞质 Fe-S 簇组装。 目标 1 是描绘 Sint 与 (Fe-S)int 的合成途径。将确定该地点 Sint 形成途径与线粒体 Fe-S 簇合成的 ISC 途径分叉。网站位于 (Fe-S)int 形成分叉也将同样被确定。将从酿酒酵母中分离线粒体 缺乏 ISC 机制组件的菌株将在线粒体-胞质混合测定中进行测试。 目标 2 是展示 Atm1 在导出 Sint 和 (Fe-S)int 方面的直接作用。我们将确定是否 中间体在 Atm1 耗尽的线粒体中积累，如果新将 Atm1 导入这些线粒体中 可以恢复导出过程。 Atm1 将被过度表达，以确定这是否会改变 Sint 和 (Fe- S)int 用于出口。导出的活性 (Fe-S)int（但不包括 Sint）可能包含结合的 GSH，这将进行测试。 目标 3 是定义细胞质中 (Fe-S)int 的初始相互作用子。我们将确定谷氧还蛋白是否 Grx3/4 和/或 Cfd1/Nbp35 支架复合物与纯化的 (Fe-S)int 相互作用，构建自己的 Fe-S 簇。 目标 4 是纯化和表征从线粒体输出的 Sint 和 (Fe-S)int。出口中间体将 通过尺寸排阻色谱法进行纯化，活性组分将通过 ICP-MS、ESI-MS 进行表征， 和其他光谱研究，旨在确定中间体的化学成分。 目标 5 是确定从哺乳动物细胞系（CAD 或 HEK293）分离的线粒体是否也产生 并导出两种不同的中间体，Sint 和 (Fe-S)int，以及它们是否可以替换为酵母 功能测定中的等效物。缺乏 ABCB7（人类 Atm1）的线粒体将接受测试 能够/不能够生成 Sint 和 (Fe-S)int 中间体并将其导出用于细胞质使用。 这项工作的重要性源于下游过程的关键性质 出口中间体。胞质 tRNA（Sint 下游）的硫醇化作用受损会导致蛋白质破坏 合成。细胞质 Fe-S 簇组装（(Fe-S)int 下游）受损可能会造成细胞灾难 由于基因组不稳定。了解 Sint 线粒体生产和输出的机制细节 和 (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。