IRON TRAFFICKING IN SACCHAROMYCES CEREVISIAE

描述（由申请人提供）：在大多数真核细胞（包括酵母和人类）中，必需的含铁辅助因子，血红素和Fe-S簇，在线粒体内合成。既然线粒体内膜必须是离子不可渗透的，就产生了区隔问题——铁是如何穿过线粒体膜的？我们鉴定了线粒体载体家族的蛋白质突变体，这些突变体对铁的分布表现出主要的和对比的影响。Mrs3/mrs4双突变体以线粒体为代价在细胞质中积累铁；Yhm1突变体表现出相反的模式，以牺牲细胞质为代价在线粒体中积累铁。在我们的第一个目标中，我们将研究这些线粒体载体的功能丧失或过表达对线粒体铁进出口的影响。最初的研究将是间接的，强调对细胞铁摄取，铁分配和铁蛋白在不同细胞区室中的地位的影响。位点定向突变的分析将把载体蛋白的关键序列基序与细胞表型相关联。在第二个目标中，已经开发了一种更直接的测定铁进入线粒体用于血红素合成的方法，并且正在开发铁运输（进出）的其他测定方法；这些将用于评估这些转运蛋白在原位通透化细胞中的转运功能。最终目标将是找到参与铁从细胞质转移到线粒体的其他基因/蛋白质。将对导致铁摄取失调的突变进行全基因组筛选。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转运途径的新成分。线粒体的组织与人类高度保守，线粒体载体蛋白，包括Mrs3/4和Yhm1具有人类同源物。因此，这些研究将对铁稳态发挥作用的人类疾病，如贫血和神经退行性变具有启示意义。

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.