Date of Award
Campus Access Dissertation
Chemistry and Biochemistry
Caryn E Outten
The transcription of iron uptake and storage genes in S. cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins, Grx3 and Grx4. The crystal structure of the thioredoxin-like domain of Grx3 has been determined at 1.5 ├â┬à resolution and represents the first published structure of this domain for the monothiol glutaredoxin family. To characterize the components of the iron regulation signaling pathway in vitro, yeast Grx3 was overexpressed in E. coli. Analytical and spectroscopic data indicate that yeast Grx3 and its paralog Grx4 form [2Fe-2S]-bridged homodimers with the conserved Grx domain active site Cys and GSH as Fe-S cluster ligands.
We have also co-expressed recombinant Grx3 with Fra2, another key player in the Aft1 iron regulation pathway. We have shown that co-expression of recombinant Grx3 with Fra2 in E. coli allows purification of a stable [2Fe-2S] cluster-containing Fra2-Grx3 heterodimeric complex. We have identified the amino acid residues in both Fra2 and Grx3 that are important for cluster binding and complex formation by mutagenesis and spectroscopic analyses. Our mutagenesis data indicate that Cys176 and the GSH-binding pocket in Grx3 are required for Fe-S cluster binding in Grx3-Fra2 heterodimers in addition to Grx3 homodimers. Cys66 and His103 in Fra2 are important for forming a stable [2Fe-2S]-bridged heterodimeric complex with Grx3, although the cluster is still intact when mutating these two amino acids individually.
Aft2(1-204), a homolog of Aft1, with a zinc-finger DNA binding domain and a CxC putative iron binding region was purified and tested for Fe-S cluster binding. When subjected to Fe-S cluster reconstitution, our preliminary evidence suggests that Aft2(1-204) binds a [2Fe-2S] cluster and forms a homodimer. Mutation of either of the Cys residues from the CxC motif abolishes Fe-S cluster binding, suggesting that the cluster may have all-cysteinyl ligation. Previous studies indicate that the CxC motif is required for interaction with Grx3/4 and for Aft1/2 function in vivo. In addition, mutation of Fe-S binding motifs in either Grx3 or Aft1/2 disrupts yeast iron homeostasis, suggesting that Fe-S cluster binding could be a link between the two proteins in their iron regulatory function.
Dingra, N. N.(2010). Characterization of the Role of Glutaredoxin 3 and Its Binding Partners In Maintaining Iron Homeostasis In Saccharomyces Cerevisiae. (Doctoral dissertation). Retrieved from http://scholarcommons.sc.edu/etd/676