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.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O₂) tension displaying a robust association in 21% O₂ that is severely diminished in 1% O₂ and contributes to O₂-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.