The mechanism of apo-target recognition in cytsolic iron sulfur cluster biosynthesis

项目总结/摘要 细胞质铁硫（FeS）簇组装（CIA）途径至关重要，因为它提供FeS簇， 对DNA复制和修复、转录和翻译至关重要的酶。尽管中情局 对于细胞生长和分裂的重要性毋庸置疑，但我们对其机制知之甚少。特别是， 缺乏关于>20种FeS-酶“靶”在其序列上如何不同的信息， 结构和功能都被确定为CIA底物，以便它们的辅因子可以在最后一步插入 的路径。最近的工作已经确定了一种多蛋白复合物，称为CIA靶向复合物（CTC）， 这对于该apo-靶识别步骤是必要的。CTC亚基，称为Met 18，Cia 1和Cia 2， 酵母，在真核生物界高度保守，它们的缺失导致FeS缺陷 辅因子成熟在胞质和核，但不是线粒体，酶。然而，了解进展 CIA的最后一步进展缓慢，部分原因是无法获得重组的体外系统， 机理分析我们最近克服了这一障碍，现在准备开始阐明 识别apo靶点的机制。反恐委员会灵活而具体地 认识到CIA目标的多样性可能是由Met 18，Cia 1和Cia 2形成多个 不同的复合物，每个复合物负责识别不同的靶标子集，通过单个靶标或 共享足以与CTC缔合的共同靶向基序的靶标子集或通过组合 这两种机制。为了揭示脱辅基蛋白靶点鉴定的分子细节，我们将1） 确定形成靶向复合物的关键残基，并评估其突变如何影响CIA 2）完成全面筛选以鉴定CTC亚基的功能残基， 精确定位，这是必要的目标结合在体外和体内;和3）阐明靶向基序利用 细胞质FeS蛋白Leu 1和核FeS蛋白Rad 3与靶向的关联 复杂.这些目标的成功完成，预计将提供有关的基本知识 靶向复合物的结构及其单个亚基的作用，揭示了驱动CIA的神秘代码 目标识别，并产生关于如何识别不同目标或目标子集的新信息， CIA系统。由于目标识别是没有很好地了解任何集群生物合成途径，该项目 有望揭示这个长期存在的黑匣子在集群生物成因领域。最后，由于缺陷 CIA功能导致对DNA损伤剂的敏感性、染色体不稳定性、延长的端粒和 其他遗传毒性表型，阐明了CIA底物识别的基本机制， 有望为理解CIA和基因组稳定性之间的关系提供必要的信息 以及CIA缺陷对人类健康和疾病的影响。

Project Summary/Abstract The cytosolic iron sulfur (FeS) cluster assembly (CIA) pathway is essential since it supplies FeS clusters to enzymes which are essential for DNA replication and repair, transcription, and translation. Despite CIA's unquestionable importance for cell growth and division, we understand little about its mechanism. In particular, there is a dearth of information regarding how the >20 FeS-enzyme “targets” differing in their sequence, structure and function are all identified as CIA substrates so that their cofactors can be inserted in the final step of the pathway. Recent work has pinpointed a multiprotein complex, termed the CIA targeting complex (CTC), as being essential for this apo-target recognition step. The CTC subunits, called Met18, Cia1 and Cia2 in yeast, are highly conserved across the eukaryotic kingdom and their depletion results in a defect in FeS cofactor maturation in cytosolic and nuclear, but not mitochondrial, enzymes. However, progress to understand the final step of CIA has been slow due, in part, to the inability to access a reconstituted in vitro system for mechanistic analysis. We have recently overcome this barrier and are now poised to begin elucidating the mechanism by which apo-targets are identified. The remarkable ability of the CTC to flexibly yet specifically recognize the diverse pool of CIA targets is likely accomplished by Met18, Cia1 and Cia2 forming multiple distinct complexes, each responsible for recognition of a distinct subset of targets, by individual targets or subsets of targets sharing a common targeting motif sufficient for association with the CTC or by a combination of these two mechanisms. To reveal the molecular details underlying apo-target identification, we will 1) identify residues critical for formation of the targeting complex and evaluate how their mutation affects CIA function in vivo; 2) complete a comprehensive screen to identify functional residues of CTC subunits and pinpoint which are essential for target-binding in vitro and in vivo; and 3) elucidate the targeting motif exploited by the cytosolic FeS protein Leu1 and the nuclear FeS protein Rad3 for their association with the targeting complex. Successful completion of these aims is expected to provide fundamental knowledge about the structure of the targeting complex and the role(s) of its individual subunits, reveal the cryptic code driving CIA target recognition, and yield new information about how different targets or subsets of targets are identified by the CIA system. Since target recognition is not well understood for any cluster biogenesis pathway, this project is expected to shed light on this long-standing black box in the cluster biogenesis field. Finally, since defects CIA function result in sensitivity to DNA damaging agents, chromosomal instabilities, elongated telomeres and other genotoxic phenotypes, elucidation of the fundamental mechanism underlying CIA substrate recognition is expected to provide information essential for understanding the relationship between CIA and genome stability and the impact of CIA deficiencies on human health and disease.