Role of redox-active thiols in the biogenesis of cytosolic and nuclear iron-sulfur proteins

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Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol and nucleus, and fulfill essential tasks in metabolism, respiration, protein translation, DNA synthesis and repair as well as in antiviral defense. Their Fe/S clusters are assembled and inserted into apoproteins by complex machinery in both mitochondria and cytosol. The cytosolic Fe/S protein assembly (CIA) machinery comprises ten known proteins which assist the maturation process in three major steps. First, a [4Fe-4S] cluster is synthesized on a scaffold complex (Cfd1-Nbp35) which requires mitochondria as a sulfur donor and an electron transfer chain for reduction. Second, the Fe/S cluster is transferred to apoproteins by Nar1 and the CIA targeting complex in a target-specific manner. Third, the Yae1-Lto1 adapter complex recruits the Fe/S protein Rli1 to the CIA targeting complex for target-specific maturation. Cia2 is a crucial component of the CIA targeting complex, and contains one of the most reactive cellular thiol groups (Cys161 in yeast) that is in the center of this project. In the ongoing funding period, we have characterized Cia2 as a general Fe/S protein biogenesis factor and spectroscopically found that Cys161 coordinates a [4Fe-4S] cluster between two Cia2 monomers. Based on these (and other) findings, we now will investigate five topics. First, we will in vitro reconstitute the assembly and dislocation pathway of the Cia2 [4Fe-4S] cluster to elucidate which other CIA factors are involved. Second, we will try to accumulate in vivo evidence for Fe/S cluster binding to Cia2, e.g., by detecting its cluster-dependent dimerization. Third, we will study whether the hyper-reactive Cys161 undergoes any other post-translational modification such as disulfide bond formation or glutathionylation, especially under conditions of low thiol reductase activity which is associated with a cytosolic Fe/S protein biogenesis defect. Forth, we will test, if bacterial Cia2 homologs are also able to bind a [4Fe-4S] cluster. Fifth, we will investigate how Cia2 establishes its target specificity in Fe/S cluster transfer and insertion. We will exploit our recent findings on the two human isoforms CIA2A and CIA2B that bind to distinct, short regions of the antiviral radical-SAM Fe/S protein viperin. Collectively, our studies will contribute to a better molecular understanding of the mechanistic roles of one of the most reactive cellular thiols in Fe/S protein biogenesis.