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: 10341169
• 负责人: ANDREW B. DANCIS
• 金额: $ 53.88万
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10341169
• 关键词: Aconitate Hydratase; Alleles; Anabolism; Apoproteins; Biogenesis; Biological Assay; Cell Line; Cell Survival; Cell physiology; Cells; Chemicals; Complex; Cysteine; Cytoplasm; Cytosol; DNA metabolism; Data; Detection; Eukaryota; Genetic; Genomic Instability; Glutathione; Homologous Gene; Human; Impairment; Individual; Inductively Coupled Plasma Mass Spectrometry; Inner mitochondrial membrane; Iron; Iron-Sulfur Proteins; Mammalian Cell; Methods; Mitochondria; Molecular Chaperones; Molecular Sieve Chromatography; Mus; Nature; Neurons; Organelles; Pathway interactions; Process; Production; Protein Biosynthesis; Protein Precursors; Proteins; Respiration; Ribosomes; Role; Saccharomyces cerevisiae; Site; Sulfur; Testing; Transfer RNA; Work; Yeasts; cofactor; cysteine desulfurase; experimental study; glutaredoxin; knock-down; mutant; 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，必需的铁-S簇辅助因子的生物合成是由线粒体分割的 铁-硫簇(ISC)机制和胞质铁-硫蛋白组装(CIA)机制。我们发现 单独的胞浆本身不能合成Fe-S团簇。然而，将线粒体添加到细胞质中，可以 这一进程将继续下去。同样，tRNAs的硫基化对于准确的蛋白质合成是至关重要的，并被分离出来 单靠胞浆不能硫化tRNA，但在添加线粒体后就会这样做。我们发现ISC 线粒体中的机械产生两种截然不同的中间产物，Sint和(Fe-S)Int。这些中间体是 通过线粒体内膜中的Atm1转运体输出到胞浆。一旦出口，Sint就是 用于tRNA硫基化和(Fe-S)INT被CIA机制用于胞内Fe-S簇组装。 目的1描述SINT和(Fe-S)INT的合成途径。将确定地点，即 Sint的形成途径与线粒体Fe-S簇合成的ISC途径分叉。该网站位于 同样将确定哪个(Fe-S)INT地层分叉。线粒体将从酿酒酵母中分离出来 缺乏ISC机器组件的菌株，它们将在线粒体-胞浆混合试验中进行测试。 目标2是证明ATM1在出口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，如果新输入的Atm1进入这些线粒体 可以恢复导出过程。Atm1将过度表达，以确定这是否会改变Sint和(Fe-)的竞争 S)国际出口。出口的活性(铁-S)INT，但不是SINT，可能含有结合的GSH，这将进行测试。 目的3确定胞浆中(Fe-S)INT的起始相互作用元件(S)。我们将确定谷氧还蛋白是否 Grx3/4和/或Cfd1/Nbp35支架复合体与纯化的(Fe-S)int相互作用，形成自己的Fe-S簇。 目的4纯化和鉴定线粒体输出的SINT和(Fe-S)INT。出口中间体将 用体积排阻色谱进行纯化，活性部位用电感耦合等离子体质谱(ICPMS)，电喷雾质谱(ESI-MS)， 和其他光谱研究，目的是确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HEK293)分离的线粒体是否也能产生 并输出两个不同的中间体，SINT和(Fe-S)INT，以及它们是否可以交换为酵母 功能分析中的等价物。缺乏ABCB7的线粒体(人类Atm1)将被检测其 有能力/无能力产生SINT和(Fe-S)INT中间体，并将其输出用于胞浆用途。 这项工作的意义来自于位于 出口中间体。胞质tRNAs(Sint下游)硫基化受损导致蛋白质中断 综合。胞内Fe-S簇组装受损((Fe-S)INT下游)可能造成细胞灾难 由于基因组的不稳定性。了解Sint线粒体产生和输出的机制细节 和(Fe-S)INT以及对这些出口物种的检测/鉴定具有非常重要的意义。