The conserved CDC motif in the yeast iron regulator Aft2 mediates iron–sulfur cluster exchange and protein–protein interactions with Grx3 and Bol2
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The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron uptake at the plasma membrane, vacuolar iron transport, and mitochondrial iron metabolism, with the net result of mobilizing iron from extracellular sources and intracellular stores. Conversely, when iron levels are sufficient, Aft1 and Aft2 interact with the cytosolic glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, which promote Aft1/2 dissociation from DNA and subsequent export from the nucleus. Previous studies unveiled the molecular mechanism for iron-dependent inhibition of Aft1/2 activity, demonstrating that the [2Fe–2S]-bridged Grx3–Bol2 heterodimer transfers a cluster to Aft2, driving Aft2 dimerization and dissociation from DNA. Here, we provide further insight into the regulation mechanism by investigating the roles of conserved cysteines in Aft2 in iron–sulfur cluster binding and interaction with [2Fe–2S]–Grx3–Bol2. Using size exclusion chromatography and circular dichroism spectroscopy, these studies reveal that both cysteines in the conserved Aft2 Cys-Asp-Cys motif are essential for Aft2 dimerization via [2Fe–2S] cluster binding, while only one cysteine is required for interaction with the [2Fe–2S]–Grx3–Bol2 complex. Taken together, these results provide novel insight into the molecular details of iron–sulfur cluster transfer from Grx3–Bol2 to Aft2 which likely occurs through a ligand exchange mechanism. Loss of either cysteine in the Aft2 iron–sulfur binding site may disrupt this ligand-exchange process leading to the isolation of a trapped Aft2–Grx3–Bol2 intermediate, while the replacement of both cysteines abrogates both the iron–sulfur cluster exchange and the protein–protein interactions between Aft2 and Grx3–Bol2.
KeywordsIron–sulfur cluster Iron regulation Glutaredoxin Circular dichroism Zinc-finger domain Glutathione Yeast
Size exclusion chromatography
This work was supported by Grant R35 GM118164 to C.E.O. from the National Institute of General Medical Sciences.