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.

摘要 在真核生物中，必需的Fe-S簇辅因子的生物合成是由线粒体的 铁-硫簇（ISC）机制和细胞溶质铁-硫蛋白组装（CIA）机制。我们发现 分离的胞质溶胶本身不能合成Fe-S簇。然而，将线粒体添加到胞质溶胶中， 这一进程继续下去。类似地，tRNA的巯基化对于精确的蛋白质合成是至关重要的，并且被分离。 单独的胞质溶胶不能使tRNA巯基化，但在加入线粒体时会这样做。我们发现ISC 线粒体中的机械产生两种不同的中间体，Sint和（Fe-S）int。 通过线粒体内膜中的Atm 1转运蛋白输出到胞质溶胶。一旦出口，Sint 用于tRNA硫醇化，并且（Fe-S）int被CIA机器用于细胞溶质Fe-S簇组装。 目的1是描绘合成Sint与（Fe-S）int的途径。 Sint形成的途径从线粒体Fe-S簇合成的ISC途径分叉。现场的球迷 同样地，将确定（Fe-S）中间形成分叉。线粒体将从S.酿酒酵母 缺乏ISC机制的组分的菌株，并且将在细胞质-细胞质混合测定中对其进行测试。 目的2是证明Atm 1在导出Sint和（Fe-S）int中的直接作用。 中间产物在Atm 1耗尽的线粒体中积累，如果新输入的Atm 1进入这些线粒体， 可以恢复出口过程。Atm 1将过表达，以确定这是否改变了Sint和（Fe-）的竞争 S）int用于导出。导出的活性（Fe-S）int（而非Sint）可能含有结合GSH，将对其进行检测。 目的3确定胞质中（Fe-S）int的初始相互作用物。我们将确定谷氧还蛋白 Grx 3/4和/或Cfd 1/Nbp 35支架复合物与纯化的（Fe-S）int相互作用以构建它们自己的Fe-S簇。 目的4：纯化线粒体输出的Sint和（Fe-S）int，并对其进行表征。出口的中间体将 通过尺寸排阻色谱法纯化，并通过ICP-MS，ESI-MS， 和其他光谱研究，目的是鉴定中间体的化学组成。 目的5是确定从哺乳动物细胞系（CAD或HEK 293）分离的线粒体是否也产生 并输出两种不同的中间体，Sint和（Fe-S）int，以及它们是否可以交换酵母 功能测定中的等同物。将测试缺乏ABCB 7（人Atm 1）的线粒体的 能够/不能产生Sint和（Fe-S）int中间体并将它们输出用于胞质溶胶。 这项工作的重要性来自下游过程的关键性质， 出口中间体。胞质tRNA（Sint下游）的巯基化受损导致蛋白质 合成.受损的胞质Fe-S簇组装（（Fe-S）int下游）可能会造成细胞灾难 因为基因组不稳定。了解线粒体生产和输出Sint的机制细节 和（Fe-S）int沿着与检测/鉴定这些输出的物种，在这里提出的是非常重要的。