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Elucidating Molecular Mechanisms of Iron-Sulfur Protein Maturation Mediated by the Cytosolic Iron-Sulfur Cluster Assembly Pathway


Iron-sulfur (Fe-S) proteins are proteins containing the omnipresent Fe-S clusters as cofactors. Studies have accumulated demonstrating that Fe-S proteins are involved in a plethora of essential cellular functions. In eukaryotes, the cytosolic iron-sulfur cluster assembly (CIA) pathway, which depends on the mitochondrial iron-sulfur cluster assembly (ISC) pathway, facilitates Fe-S cluster incorporation into extramitochondrial Fe-S proteins. These include nuclear proteins required for DNA replication and DNA damage repair, as well as cytosolic proteins required for maintaining cellular iron homeostasis and ribosomal functions. In the CIA pathway, [4Fe-4S] cluster are assembled on the CIA scaffold complex, transferred to CIAO3, and incorporated into CIA substrates via the CIA targeting complex. The maturation of CIA substrates is controlled by cellular iron and oxygen. We demonstrate in this study that the incorporation of CIAO3 into CIA machineries is iron regulated, which may account for this precise control of substrate maturation. We developed a targeted proteomics assay to monitor the presence and abundance of known CIA components and prototypical substrates. Using this assay, we were able to detect that the CIA targeting complex and CIA substrates associated with NUBP2, a component of the CIA scaffold complex. This suggests the possible formation of higher order meta complexes composed of the CIA scaffold complex, CIAO3, the CIA targeting complex and CIA substrates. We show that the interaction between CIAO3 and the CIA scaffold complex is affected by cellular iron availability, and this interaction is additionally strengthened under hypoxic environments and weakened by reactive oxygen species. Furthermore, we found that CIAO3 integration into CIA machineries demands a functional ISC pathway. Moreover, we generated CIAO3 mutants defective in Fe-S cluster binding and observed reduced interactions with both the CIA scaffold complex and the CIA targeting complex. However, stronger interactions with substrates were observed in these mutants, suggesting that CIAO3 and CIA substrates may be present in complexes in the absence of the CIA targeting complex. Lastly, we revealed that the CIAO3 mutant that associates with pulmonary arteriovenous malformations is incapable of integrating into the CIA machineries, which may partially explain the pathological outcome of this mutation. Together, these findings demonstrate the reorganization of the CIA machinery in different cellular environments. Alongside this, we investigated the architecture of the CIA targeting complex with crosslinking mass spectrometry and found that CIAO2B is in contact with the C-terminus of MMS19. A CIA substrate, CDKAL1, is also in close proximity to the C-terminus of MMS19.

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