Mitochondria-cytoplasm interactions for cytosolic Fe-S cluster assembly

描述（由申请人提供）：铁硫（Fe-S）簇是包括tRNA硫代化在内的许多细胞过程所需的必需蛋白质辅因子。在酵母和人类中，Fe-S蛋白存在于线粒体和细胞质中。线粒体中的一个特殊系统，称为铁硫团簇（ISC）机制，催化铁硫团簇合成，并且分离的线粒体本身可以形成团簇。Fe-S簇在胞浆中的组装需要细胞质铁硫蛋白组装（CIA）机制。然而，中央情报局的系统并不是单独工作的。我们假设线粒体中的ISC机制产生含硫中间体（Sint），该中间体由atp依赖性转运体Atm1输出，并被细胞质中的CIA用于Fe-S簇合成和tRNA硫代化。在使用35s半胱氨酸作为硫供体的新实验中，我们发现分离的细胞质不能合成Fe-S簇或硫代trna。然而，在细胞质中加入线粒体或线粒体产生的硫物质允许这些过程发生。目的1是确定酵母中Sint形成和使用的要求-线粒体产生它，细胞质使用它进行Fe-S簇组装和tRNA硫代化。实验将涉及线粒体Fe-S簇合成的操作，以及线粒体或细胞质溶胶中核苷酸的单独操作。目的2是定义Atm1及其输出底物的功能。在我们的实验中，完整的atm1突变体线粒体不支持细胞质Fe-S簇组装或tRNA硫代化。实验将确定这些过程是否可以通过完整的atm1线粒体和新输入的atm1，或通过破坏线粒体膜绕过atm1的输出阻断来恢复。Atm1出口的活性Sint物种将通过色谱纯化、质谱分析和其他生物物理方法进行鉴定。目的3是确定铁的来源和Dre2在酵母细胞质中Fe-S簇合成中的作用。实验将确定铁的来源细胞质簇组装-线粒体或细胞质。Dre2是一种重要的CIA成分，它与Tah18形成还原酶复合物。纯化的Dre2 - Tah18复合体在缺乏Dre2（或Tah18）的细胞质中恢复Fe-S簇组装的能力将被检验。目的4是明确ABCB7（人Atm1）和CIAPIN1（人Dre2）在哺乳动物细胞胞质Fe-S簇组装和凋亡中的功能。这些蛋白可能通过影响胞质Fe-S簇组装来发挥其抗凋亡功能，这一假设将得到验证。Fe-S簇生物发生是保守的，上述所有蛋白在人和酵母中都有同源物。Fe-S簇组装紊乱导致骨髓衰竭、神经变性和肌病等疾病表现。在与ABCB7功能障碍相关的铁母细胞贫血中，红细胞前体积累了毒性量的线粒体铁，并发生凋亡。线粒体-细胞质相互作用是导致这种疾病和其他人类疾病的核心原因。

DESCRIPTION (provided by applicant): Iron-sulfur (Fe-S) clusters are essential protein cofactors required for numerous cellular processes including tRNA thiolation. In yeast and humans, Fe-S proteins are found in mitochondria and cytoplasm. A specialized system in mitochondria, called the Iron-Sulfur Cluster (ISC) machinery, catalyzes Fe-S cluster synthesis, and isolated mitochondria by themselves can form clusters. Fe-S cluster assembly in cytosol requires the Cytoplasmic Iron-Sulfur Protein Assembly (CIA) machinery. However, the CIA system does not work by itself. We hypothesize that the ISC machinery in mitochondria generates a sulfur-containing intermediate (Sint), which is exported by the ATP-dependent transporter Atm1 and used by the CIA in the cytosol for Fe-S cluster synthesis and tRNA thiolation. In novel assays using 35S-cysteine as the sulfur donor, we find that isolated cytoplasm cannot synthesize Fe-S clusters or thiolate tRNAs. However, addition of mitochondria or a mitochondrial generated sulfur species to the cytoplasm allows these processes to occur. Aim 1 is to define the requirements for formation and use of Sint in yeast - mitochondria produce it and cytoplasm uses it for Fe-S cluster assembly and tRNA thiolation. Experiments will involve manipulations of mitochondrial Fe-S cluster synthesis, and separate manipulations of nucleotides in mitochondria or cytosol. Aim 2 is to define the function of Atm1 and its export substrate. Intact atm1 mutant mitochondria did not support cytoplasmic Fe-S cluster assembly or tRNA thiolation in our assays. Experiments will determine if these processes can be restored by intact atm1 mitochondria with newly imported Atm1, or by bypassing the export block in atm1 through disruption of mitochondrial membranes. The active Sint species exported by Atm1 will be identified by chromatographic purifications, mass spectrometry, and other biophysical methods. Aim 3 is to define the source of iron and the role of Dre2 in Fe-S cluster synthesis in yeast cytoplasm. Experiments will determine the origin of iron for cytosolic cluster assembly - mitochondria or cytoplasm. Dre2 is an essential CIA component, and it forms a reductase complex with Tah18. The ability of purified Dre2�Tah18 complex to restore Fe-S cluster assembly in cytoplasm lacking Dre2 (or Tah18) will be examined. Aim 4 is to define the functions of ABCB7 (human Atm1) and CIAPIN1 (human Dre2) in cytoplasmic Fe-S cluster assembly and apoptosis in mammalian cells. These proteins might perform their anti-apoptotic functions via effects on cytosolic Fe-S cluster assembly, and this hypothesis will be tested. Fe-S cluster biogenesis is conserved, and all of the proteins mentioned above have orthologs in humans and yeast. Perturbed Fe-S cluster assembly results in disease manifestations such as bone marrow failure, neurodegeneration and myopathy. In sideroblastic anemia associated with ABCB7 dysfunction, red cell precursors accumulate toxic amounts of mitochondrial iron, and they undergo apoptosis. Mitochondria- cytoplasm interactions are central to causation of this and other human diseases.