Biochemistry and genetics of iron transport in mitochondria and related processes

描述（由申请人提供）：在大多数真核细胞（包括酵母和人类）中，用于合成血红素的铁插入步骤仅发生在线粒体内。同样，用于形成新的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（维持生电膜电位的要求），因此出现了血红素和FeS簇中的铁如何进入线粒体的问题。来自几个实验室的最新数据表明，线粒体载体蛋白Mrs 3和Mrs 4参与酵母，直系同源物mitoferrin 1和2在脊椎动物。然而，铁运输过程的许多方面仍然未知。Frataxin是一种小的保守的线粒体蛋白，参与线粒体内血红素和FeS簇的铁利用下游，但其确切功能也不清楚。在这里，我们提出了三个遗传和生物化学的目标，以进一步深入研究这一过程。目的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Mrs 3和Mrs 4的作用以及产电膜电位在介导铁转运中的作用。将评价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，并研究含有转运蛋白的约660 kDa的巨大蛋白复合物的作用。目的2）确定frataxin在线粒体内FeS和血红素合成中的作用。线粒体中共济失调蛋白的量将在很大范围内变化，靶向突变将被分析为与铁、Isu1（FeS簇组装支架）和亚铁螯合酶（血红素合成酶）相互作用位点中的特定突变。在短时间范围内对分离的线粒体进行的测定将使直接效应与继发效应区分开来。在目标3）中，将通过正在进行的筛选来寻找与铁转运到线粒体相关的新基因和蛋白质，以鉴定mrs 3和mrs 4的合成致死突变。早期的结果表明，在该筛选中鉴定的单个突变体（例如dre2，tsa1）参与铁代谢。铁转运进入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。参与铁运输和线粒体中使用的特定酵母蛋白具有人类直系同源物（共济失调蛋白，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，因此酵母研究的结果将与人类疾病有关。

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.