Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Caryn E Outten

Abstract

To maintain optimal intracellular iron levels, iron transport and storage is tightly regulated in all eukaryotic cells ranging from yeast to humans. However, there are significant gaps in our understanding of iron sensing and regulation mechanisms at the cellular and molecular level. To address these gaps, we are employing complementary approaches to characterize the structural and functional interactions of the monothiol glutaredoxins (Grxs) and BolA-like protein families that have recently emerged as novel players in iron signaling.

The model eukaryote Saccharomyces cerevisiae (bakers' yeast) has provided some important insights into the molecular functions of these two protein families. Transcription of iron uptake and storage genes in yeast is regulated by the transcription factor Aft1 and its paralogue Aft2. Nucleocytoplasmic shuttling of Aft1 (and presumably Aft2) is dependent upon a signaling pathway that includes the cytosolic monothiol glutaredoxins, Grx3 and Grx4, and the BolA-like protein Fra2. Our published results demonstrate that Fra2 and Grx3/4 form an unusual [2Fe-2S]2+-bridged heterodimer with Fe ligands provided by a cysteine from Grx3/4, a histidine from Fra2, and the tripeptide glutathione (GSH), which are all critical for iron-dependent regulation of Aft1/2 activity in vivo. To further unravel the regulation mechanism of Aft1/2 activity in response to iron, we have purified Aft2 and demonstrated that Aft2 specifically interacts with the [2Fe-2S] Fra2-Grx3 heterodimer through its Cys-x-Cys motif, but does not interact with apo-Fra2, apo-Grx3, or the [2Fe-2S]-bridged Grx3 homodimer. Dissection of this interaction reveals cluster transfer from Fra2-Grx3 to Aft2, leading to Aft2 dimerization. Collectively, these data suggest a regulation model in which the [2Fe-2S] Fra2-Grx3/4 complex acts as a cellular iron sensor that specifically interacts with the transcriptional regulators Aft1 and Aft2 under iron-replete conditions and stimulates their dimerization by cluster transfer. Dimerization, in turn, may be more favorable for their cytosolic localization, thus deactivating expression of iron uptake and storage genes in yeast.

In addition, [2Fe-2S] Fra2-Grx3 homologues from E. coli and human were also characterized. Biophysical studies demonstrate that yeast and human Grx-BolA complexes have similar cluster coordination environments, whereas E. coli Grx4-BolA complexes are distinct from their eukaryotic homologues. Overall, it suggests that the clusters on Grx-BolA complexes in eukaryotes possess different characteristics from those in prokaryotes, although complex formation between Grxs and BolA-like proteins are well conserved throughout evolution.

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