Vashisht Ajay A, Yu Clarissa C, Sharma Tanu, Ro Kevin, Wohlschlegel James A
From the Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, California 90095.
From the Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, California 90095
J Biol Chem. 2015 May 29;290(22):14218-25. doi: 10.1074/jbc.M115.650762. Epub 2015 Apr 20.
Xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH (TFIIH) transcription complex and plays essential roles in transcription and nucleotide excision repair. Although iron-sulfur (Fe-S) cluster binding by XPD is required for activity, the process mediating Fe-S cluster assembly remains poorly understood. We recently identified a cytoplasmic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, CIAO1, and FAM96B that is required for the biogenesis of extramitochondrial Fe-S proteins including XPD. Here, we use XPD as a prototypical Fe-S protein to further characterize how Fe-S assembly is facilitated by the CIA targeting complex. Multiple lines of evidence indicate that this process occurs in a stepwise fashion in which XPD acquires a Fe-S cluster from the CIA targeting complex before assembling into TFIIH. First, XPD was found to associate in a mutually exclusive fashion with either TFIIH or the CIA targeting complex. Second, disrupting Fe-S cluster assembly on XPD by either 1) depleting cellular iron levels or 2) utilizing XPD mutants defective in either Fe-S cluster or CIA targeting complex binding blocks Fe-S cluster assembly and prevents XPD incorporation into TFIIH. Finally, subcellular fractionation studies indicate that the association of XPD with the CIA targeting complex occurs in the cytoplasm, whereas its association with TFIIH occurs largely in the nucleus where TFIIH functions. Together, these data establish a sequential assembly process for Fe-S assembly on XPD and highlight the existence of quality control mechanisms that prevent the incorporation of immature apoproteins into their cellular complexes.
着色性干皮病D组（XPD）解旋酶是转录因子IIH（TFIIH）转录复合物的一个组成部分，在转录和核苷酸切除修复中发挥着重要作用。虽然XPD结合铁硫（Fe-S）簇是其活性所必需的，但介导Fe-S簇组装的过程仍知之甚少。我们最近鉴定出一种由MMS19、CIAO1和FAM96B组成的细胞质Fe-S簇组装（CIA）靶向复合物，它是包括XPD在内的线粒体外Fe-S蛋白生物合成所必需的。在这里，我们以XPD作为典型的Fe-S蛋白，进一步表征CIA靶向复合物如何促进Fe-S组装。多条证据表明，这个过程以逐步的方式发生，其中XPD在组装到TFIIH之前从CIA靶向复合物中获得一个Fe-S簇。首先，发现XPD以互斥的方式与TFIIH或CIA靶向复合物结合。其次，通过以下两种方式破坏XPD上的Fe-S簇组装：1）降低细胞铁水平或2）利用在Fe-S簇或CIA靶向复合物结合方面有缺陷的XPD突变体，会阻止Fe-S簇组装并防止XPD掺入TFIIH。最后，亚细胞分级分离研究表明，XPD与CIA靶向复合物的结合发生在细胞质中，而它与TFIIH的结合主要发生在TFIIH发挥功能的细胞核中。总之，这些数据确立了XPD上Fe-S组装的顺序组装过程，并突出了质量控制机制的存在，这些机制可防止未成熟的脱辅基蛋白掺入其细胞复合物中。
