Biochemistry and genetics of iron transport in mitochondria and related processes

线粒体铁转运及相关过程的生物化学和遗传学

• 批准号: 7891077
• 负责人: ANDREW B. DANCIS
• 金额: $ 10.1万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7891077
• 关键词: Anemia; Binding Sites; Biochemical; Biochemistry; Biological Assay; Carrier Proteins; Categories; Data; Defect; Enzymes; Eukaryota; Eukaryotic Cell; Gene Proteins; Generations; Genetic; Goals; Hand; Heme; Heme Iron; Human; Inner mitochondrial membrane; Ions; Iron; Mediating; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Mutation; Nerve Degeneration; Orthologous Gene; Porphyrias; Process; Proteins; Role; Site; System; Time; Transport Process; Vertebrates; Yeasts; ferrochelatase; frataxin; human disease; iron metabolism; mutant; novel; protein complex; scaffold; uptake; yeast protein

DESCRIPTION (provided by applicant): In most eukaryotic cells (including yeast and humans), the iron insertion step for synthesis of heme occurs exclusively within mitochondria. Likewise, iron insertion for formation of new FeS clusters can occur within mitochondria. Since the mitochondrial inner membrane is impermeable to ions (a requirement for maintaining the electrogenic membrane potential), the question arises of how iron for heme and FeS clusters gets into mitochondria. Recent data from several labs have demonstrated involvement of mitochondrial carrier proteins Mrs3 and Mrs4 in yeast, and orthologs mitoferrin 1 and 2 in vertebrates. However, many aspects of the iron transport process remain unknown. Frataxin a small conserved mitochondrial protein is involved downstream in iron use for heme and FeS clusters within mitochondria, but its precise function is also unclear. Here we propose three genetic and biochemical aims to delve further into this process. Aim 1) will seek to characterize the iron transport process in isolated yeast mitochondria in short time frames. The role of Mrs3 and Mrs4, and the role of the electrogenic membrane potential in mediating iron transport will be studied. Targeted mutations in the transporters, specifically in the hypothetical substrate binding site, will be evaluated, and the role of a giant protein complex of roughly 660 kDa containing the transporter(s) will be investigated. Aim 2) will ascertain the role of frataxin in FeS and heme synthesis within mitochondria. The amount of frataxin in mitochondria will be varied over a large range and targeted mutations will be analyzed specifically mutations in interaction sites with iron, with Isu1 (scaffold for FeS cluster assembly) and with ferrochelatase (enzyme for heme synthesis). Assays in isolated mitochondria in short time frames will allow direct effects to be distinguished from secondary effects. In aim 3), novel genes and proteins connected to iron transport to mitochondria will be sought by means of an ongoing screen to identify mutations that are synthetically lethal with mrs3 and mrs4. Early results show that single mutants (e.g. dre2, tsa1) identified in this screen are involved in iron metabolism. Iron transport into mitochondria is an essential conserved process in eukaryotes, required for heme synthesis and FeS cluster assembly. Proposed studies will be performed in yeast mitochondria, but the organization of mitochondria is highly conserved between yeast and humans. The particular yeast proteins involved in iron transport and use in mitochondria have human orthologs (frataxin, mitochondrial carriers). Defects in these proteins have been implicated in porphyria, anemia and neurodegeneration, and so results obtained in with yeast will be relevant to human disease.

描述（由申请人提供）：在大多数真核细胞（包括酵母和人类）中，合成血红素的铁插入步骤仅发生在线粒体内。同样，用于形成新 FeS 簇的铁插入可以发生在线粒体内。由于线粒体内膜对离子是不可渗透的（这是维持生电膜电位的必要条件），因此出现了血红素和 FeS 簇中的铁如何进入线粒体的问题。多个实验室的最新数据表明，酵母中线粒体载体蛋白 Mrs3 和 Mrs4 以及脊椎动物中的直系同源物线粒体铁蛋白 1 和 2 都参与其中。然而，铁运输过程的许多方面仍然未知。 Frataxin 是一种小型保守的线粒体蛋白，参与线粒体内血红素和 FeS 簇的铁利用下游，但其确切功能尚不清楚。在这里，我们提出了三个遗传和生化目标来进一步深入研究这一过程。目标 1) 将寻求在短时间内表征分离的酵母线粒体中的铁转运过程。将研究 Mrs3 和 Mrs4 的作用以及生电膜电位在介导铁转运中的作用。将评估转运蛋白中的靶向突变，特别是假设的底物结合位点中的突变，并将研究包含转运蛋白的约 660 kDa 的巨型蛋白质复合物的作用。目标 2) 将确定 frataxin 在线粒体内 FeS 和血红素合成中的作用。线粒体中 frataxin 的量将在很大范围内变化，并且将专门分析与铁、Isu1（用于 FeS 簇组装的支架）和亚铁螯合酶（用于血红素合成的酶）相互作用位点的突变。在短时间内对分离的线粒体进行测定将能够区分直接效应和次要效应。在目标 3) 中，将通过持续筛选来寻找与铁转运至线粒体相关的新基因和蛋白质，以识别对 mrs3 和 mrs4 具有综合致死性的突变。早期结果表明，本次筛选中鉴定出的单个突变体（例如 dre2、tsa1）参与铁代谢。铁转运到线粒体是真核生物中一个重要的保守过程，是血红素合成和 FeS 簇组装所必需的。拟议的研究将在酵母线粒体中进行，但线粒体的组织在酵母和人类之间高度保守。参与铁转运和线粒体使用的特定酵母蛋白具有人类直系同源物（frataxin、线粒体载体）。这些蛋白质的缺陷与卟啉症、贫血和神经变性有关，因此酵母获得的结果将与人类疾病相关。