The cytosolic iron sulfur cluster assembly (CIA) system is a highly conserved pathway that assembles and inserts iron sulfur (FeS) cluster cofactors into cytosolic and nuclear proteins. These CIA targets are involved in many important cellular processes including DNA replication and repair, iron homeostasis, and translation. The mechanism by which the CIA substrates, numbering more than 25 proteins, are identified in the last step of the pathway is not understood. However, it is known that the CIA proteins Met18, Cia1 and Cia2 form a large complex, called the CIA targeting complex, which appears to be responsible for substrate recognition in the final step of the pathway. In this work, we examined how Rad3, a DNA helicase which binds a Fe4S4 cluster, associates with two subunits of the CIA targeting complex, Met18 and Cia2. Site directed mutagenesis combined with affinity co-purification assays were utilized to investigate a 10 amino acid region that, when deleted, prevents cofactor acquisition by Rad3 in vivo. To pinpoint which amino acids are critical for recognition of Rad3, site-directed mutants of Rad3 were tested for their ability to bind Met18-Cia2. Deletion of the 10 amino acid region prevented binding of Rad3 in vitro. Although alanine scanning mutagenesis suggested that 3 of the 10 amino acids are required for interaction, our recent results suggest that observation can be explained by a destabilizing effect on tertiary structure. These results indicate that the recognition of Rad3 by the targeting complex depends on the tertiary structure of Rad3 and rule out the possibility that Rad3 is recognized by a short, linear motif. It appears that none of the previously identified 10 amino acids are directly involved with binding to the CIA targeting complex and that the complex recognizes the tertiary structure of a domain far from the FeS cluster binding site.

Characterizing the binding of Rad3 to the cytosolic iron sulfur cluster assembly targeting complex

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.