The conserved CDC motif in the yeast iron regulator Aft2 mediates iron-sulfur cluster exchange and protein-protein interactions with Grx3 and Bol2

Abstract

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.

Keywords: Circular dichroism; Glutaredoxin; Glutathione; Iron regulation; Iron–sulfur cluster; Yeast; Zinc-finger domain.

Figure 1.

Titration of [2Fe-2S] Grx3-Bol2 with apo-Aft2 monitored by UV-visible CD spectroscopy. CD spectra for [2Fe-2S]²⁺ cluster-bound Grx3-Bol2 heterodimers (black line) titrated with 0.25–5-fold excess Aft2(WT) (a), Aft2(C187A) (b), or Aft2(C189A) (c) (gray lines). Colored lines in each graph (blue, pink, purple) correspond to 5-fold excess Aft2 added. Δɛ values are based on the [2Fe-2S]²⁺ cluster concentration (40 μM). d Difference in CD intensity between 400 and 473 nm (arrows in a) as a function of [Aft2] from CD spectra shown in (a-c).

Figure 2.

Top, Size exclusion chromatograms of Aft2(WT), Aft2 Cys variants, and [2Fe-2S] Grx3-Bol2 run separately. Bottom, Size exclusion chromatograms of Aft2(WT) or Aft2 Cys variants mixed with [2Fe-2S] Grx3-Bol2 in a 2:1 ratio prior to loading. SDS-PAGE analysis of fractions collected from each chromatography run are shown in Supplementary Fig. 1.

Figure 3.

SDS-PAGE analysis following heparin chromatography separation of Aft2(WT) or [2Fe-2S]-Grx3-Bol2 alone, or Aft2(WT) or CDC variants mixed in a 2:1 ratio with [2Fe-2S]-Grx3-Bol2. The % Fe measured in the flow-through (F-T) vs. the eluate (E) for each mixture is shown below each lane. MW std, protein molecular weight standards; Fe-S, [2Fe-2S]-Grx3-Bol2. Aft2 = 23.3 kDa, Grx3 = 28.1 kDa, Bol2 = 14.0 kDa. Bol2 typically runs as two bands on the gel due to partial proteolysis [14,16].

Figure 4.

(a) Top, Size exclusion chromatograms of eluates after heparin column separation for 2:1 mixtures of Aft2 variants + [2Fe-2S]-Grx3-Bol2. Bottom, SDS-PAGE analysis of the fractions collected for each mixture. (b) UV-visible absorption (top) and CD (bottom) spectra of heparin column eluates for 2:1 mixtures of Aft2 variants + [2Fe-2S] Grx3-Bol2 (colored solid lines), as compared to [2Fe-2S]-Grx3-Bol2 alone (black dashed line). Black, vertical dotted lines correspond to major UV-visible absorption peaks in [2Fe-2S]-Grx3-Bol2. The red vertical dotted lines and red arrows indicate the shift in these peaks in the Aft2(WT) sample (blue spectrum). ɛ and Δɛ values are normalized to protein concentrations.

Figure 5.

Model for Fe-S cluster transfer between [2Fe-2S] Grx3-Bol2 and Aft2. Bol2 facilitates the interaction with Aft2 allowing access to the [2Fe-2S] cluster. Cys187 in Aft2 (in red) may bind at or near the cluster in the ternary [2Fe-2S] cluster-bound intermediate complex, displacing a ligand from Grx3, Bol2 or GSH (2 of these possible conformations are shown in the model). Cys189 in Aft2 and a second Aft2 molecule is required to complete the [2Fe-2S] transfer and form the [2Fe-2S]-bridged Aft2 homodimer.