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

项目摘要/摘要 胞质铁硫(FeS)簇组装(CIA)途径是必不可少的，因为它提供FeS簇到 对DNA复制和修复、转录和翻译至关重要的酶。尽管中情局 对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-酶在其序列上如何不同的“靶标”的信息， 结构和功能都被确定为CIA底物，因此它们的辅因子可以在最后一步中插入 在这条小路上。最近的研究确定了一种多蛋白复合体，称为中央情报局靶向复合体(CTC)， 对于这个非目标识别步骤来说是必不可少的。CTC亚基，称为Met18、Cia1和Cia2 酵母在真核生物王国中高度保守，它们的枯竭导致FeS的缺陷。 辅因子在胞浆酶和核酶中成熟，但在线粒体中不成熟。然而，要理解的进展 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 机械分析。我们最近已经克服了这一障碍，现在准备开始澄清 识别脱辅酶靶标的机制。反恐委员会有能力灵活而具体地 认识到CIA目标的多样性很可能是通过Met18、Cia1和Cia2形成多个 不同的复合体，每个负责识别不同的目标子集，由单个目标或 目标子集共享足以与反恐委员会联系或通过其组合的共同目标主题 这两种机制中。为了揭示脱辅酶靶标识别的分子细节，我们将1) 确定对形成靶向复合体至关重要的残基，并评估其突变如何影响CIA 2)完成对CTC亚基功能残基的全面筛选，并 对体外和体内靶向结合是必不可少的；以及3)阐明所利用的靶向基序 胞质FES蛋白Leu1和核FES蛋白Rad3与靶向的关系 很复杂。这些目标的成功完成预计将提供关于 靶向复合体的结构及其各个亚基的作用(S)，揭示了推动CIA的神秘代码 目标识别，并产生关于不同目标或目标子集如何被 中情局的系统。由于对于任何集群生物发生途径都不能很好地理解目标识别，本项目 有望揭开星系团生物发生领域中这个长期存在的黑匣子的面纱。最后，由于缺陷 CIA功能导致对DNA损伤剂、染色体不稳定性、延长的端粒和 其他遗传毒性表型，阐明CIA底物识别的基本机制是 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.