| Description |
Iron-sulfur (Fe/S) clusters are essential cofactors required to perform unique functions in nitrogen fixation, respiration, metabolite catabolism, ribosome assembly, DNA repair, etc., through their redox-active characteristic, the ability to accept or donate an additional election. Biology has evolved intricate mechanisms to assemble and subsequently transfer the preformed Fe/S clusters to client proteins. Fe/S clusters are found in many of the cell’s subcompartments, but all of the clusters’ biogenesis begins in the mitochondria. The mitochondrial iron-sulfur biogenesis pathway (ISC) starts at the ISU complex with the removal of a sulfide from cysteine by a cysteine desulfurase. This sulfide is transferred to a scaffold protein where, with two irons and a reluctant, a [2Fe-2S] cluster is made. This preformed cluster is transferred from the scaffold through a gludaredoxin to the ISA complex. The ISA complex, comprised of three proteins, Isa1, Isa2, and Iba57, receives two [2Fe-2S] clusters that are then condensed into a single [4Fe-4S] cluster. This preformed [4Fe-4S] cluster must then be transferred from the assembly complex to client proteins in a manner that does not expose it to oxidative damage. The work in this dissertation focused on the biochemical characterization of Nfu1 as the transfer protein for [4Fe-4S] clusters formed by the ISC pathway. Nfu1 was shown to genetically interact with Isa2 and physically interact with all members of the ISA complex. Additionally, physical interactions between Nfu1 and all of the [4Fe-4S] clients in the yeast, Saccharomyces cerevisiae, were identified through proteomic studies. These studies went onto demonstrate the BolA family member Bol3 functions with Nfu1 in the cluster transfer process. A follow-up study of Nfu1 sought to identify how it recognized client proteins for delivery of a [4Fe-4S] cluster. Using computational approaches identified a five amino acid motif, TIMNM. Sufficiency was demonstrated by creating an interaction between Nfu1 with a heterologous chimeric protein fused with the motif. Necessity was shown in both Sdh2 and aconitase client proteins with mutations to the motif of either client resulting in defects in enzymatic activity and a loss of interaction with Nfu1. Overall, these studies demonstrate that Nfu1 transfers [4Fe-4S] clusters to client proteins that are identified by the TIMNM Nfu1 recognition motif. |
