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，铁插入血红素的插入步骤仅在线粒体内发生。同样，线粒体内可能发生用于形成新FES簇的铁插入。由于线粒体内膜不可渗透离子（维持电膜电位的要求），因此提出的问题是血红素和FES簇如何进入线粒体。来自几个实验室的最新数据表明，线粒体载体蛋白MRS3和MRS4参与酵母，以及直系同源物丝纹蛋白1和2在脊椎动物中。但是，铁运输过程的许多方面仍然未知。 Frataxin一种小的保守线粒体蛋白在线粒体内的铁质和FES簇中的下游涉及下游，但其精确功能也不清楚。在这里，我们提出了三个遗传和生化旨在进一步深入研究这一过程。 AIM 1）将寻求在短时间内孤立的酵母线粒体中的铁运输过程。将研究MRS3和MRS4的作用，以及电源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，特别是假设底物结合位点中的靶向突变，并将研究大约660 kDa的巨型蛋白质复合物的作用。 AIM 2）将确定Frataxin在FES和线粒体内血红素合成中的作用。线粒体中的Frataxin量将在较大的范围内变化，并且将在具有铁的相互作用位点特异性突变，其中ISU1（ISU1的支架（FES簇组装的支架））和铁chelatase（血红素合成酶的酶）。在短时间内进行孤立线粒体的测定将使直接效应与次要效应区分开。在AIM 3）中，将通过持续的筛选来寻求与铁运输到线粒体相关的新型基因和蛋白质，以鉴定与MRS3和MRS4合成致命的突变。早期结果表明，在此筛选中鉴定出的单个突变体（例如DRE2，TSA1）参与铁代谢。铁进入线粒体是真核生物中必不可少的保守过程，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菌和人之间是高度保守的。在线粒体中参与铁运输和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，因此用酵母获得的结果与人类疾病有关。