IRON TRAFFICKING IN SACCHAROMYCES CEREVISIAE

描述(申请人提供)：在大多数真核细胞(包括酵母和人类)中，含有必需铁辅因子的血红素和铁-S簇是在线粒体中合成的。由于线粒体内膜必须对离子不能渗透，就产生了一个分隔问题--铁是如何穿过线粒体膜的？我们鉴定了线粒体载体家族蛋白的突变体，这些突变体对铁的分布表现出显著和不同的影响。Mrs3/mrs4双突变体表现为铁在细胞质中积累，而YHM1突变体则相反，铁在线粒体中积累，而在细胞质中积累。在我们的第一个目标中，我们将研究这些线粒体载体功能丧失或过度表达对线粒体铁进出口的影响。最初的研究将是间接的，重点是对细胞铁摄取、铁分配和铁蛋白在不同细胞间隔中的状态的影响。对定点突变的分析将把载体蛋白的关键序列基序与细胞表型联系起来。在第二个目标中，已经开发了一种更直接的方法来检测线粒体对铁的吸收以合成血红素，并正在开发其他铁运输(进出)的分析方法；这些方法将被用来评估这些转运蛋白在原位通透性细胞中的运输功能。最终目标将是寻找与铁从细胞质向线粒体转移有关的其他基因/蛋白质。将进行全基因组范围的突变筛查，以寻找导致铁摄取错误的突变。Mrs3/4或YHM1与其他基因之间的合成致死关系可能揭示细胞内铁运输途径的新组成部分。线粒体的组织在人类中是高度保守的，线粒体载体蛋白，包括MRS 3/4和YHM1都有人类的同源基因。因此，这些研究将对铁稳态起作用的人类疾病产生影响，如贫血和神经退化。

DESCRIPTION (provided by applicant): In most eukaryotic cells (including yeast and humans), the essential iron containing cofactors, heme and Fe-S clusters, are synthesized within mitochondria. Since the mitochondrial inner membrane must be impermeable to ions, a compartmentation problem is created - how does iron cross the mitochondrial membrane? We identified mutants of proteins of the mitochondrial carrier family that showed major and contrasting effects on iron distribution. Mrs3/mrs4 double mutants showed iron accumulation in the cytoplasm at the expense of mitochondria; Yhm1 mutants showed an opposite pattern, with iron accumulation in mitochondria at the expense of the cytoplasm. In our first aim, we will examine the effects of loss of function or overexpression of these mitochondrial carriers on iron import and export from mitochondria. Initial studies will be indirect, emphasizing effects on cellular iron uptake, iron partitioning and the status of iron proteins in different cellular compartments. Analysis of site directed mutants will correlate critical sequence motifs of the carrier proteins with the cellular phenotypes. In the second aim, a more direct assay for uptake of iron into mitochondria for heme synthesis has been developed and additional assays of iron transport (in and out) are being developed; these will be used to assess transport functions of these transporters in permeabilized cells in situ. The final aim will be to find other genes/proteins involved in transfer of iron from cytosol to mitochondria. A genome wide screen for mutations that lead to misregulated iron uptake will be undertaken. Synthetic lethal relationships between mrs3/4 or yhm1 and other genes may reveal new components of intracellular iron trafficking pathways. The organization of mitochondria is highly conserved with humans, and mitochondrial carrier proteins, including Mrs3/4 and Yhm1 have human orthologs. Therefore these studies will have implications for human diseases in which iron homeostasis plays a role, such as anemia and neurodegeneration.