Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal

Abstract

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

Keywords: BolA-like protein; GATA-type transcription factor; circular dichroism; fission yeast; iron metabolism; iron-sulfur protein; metal homeostasis; monothiol glutaredoxin; protein-protein interaction.

Figure 1

CD-monitored titration studies of [2Fe-2S]-Grx4 and [2Fe-2S]-GRX domain with WT apo-Fra2.A, [2Fe-2S]-Grx4 (thick blue line) was titrated with increasing concentrations of Fra2 (thin black lines). A 25-fold excess Fra2 is shown as a thick purple line. B, [2Fe-2S]-GRX domain (thick green line) was titrated with a 0.125- to 3-fold excess of Fra2 (thin black lines). A 3-fold excess Fra2 is shown as a thick purple line. The arrows at selected wavelengths indicate the direction of intensity change with increasing Fra2 concentration. Δε values are based on the [2Fe-2S] cluster concentration (50 μM for [2Fe-2S]-Grx4 and 60 μM for [2Fe-2S]-GRX domain). C, the percent CD intensity change (between 408 and 458 nm) for spectra in A and B was plotted as a function of the Fra2:[2Fe-2S] ratio.

Figure 2

Size-exclusion chromatography analysis of GRX domain-Fra2 interactions. Top, chromatograms of apo-Fra2 (light blue line), apo-GRX domain (black line), and 1:1 mixture of apo-GRX domain + apo-Fra2 (dark blue line). Middle, [2Fe-2S] GRX domain (pink lines). Bottom, 1:1 mixture of [2Fe-2S]-GRX domain + apo-Fra2 (purple lines). All solid line chromatograms are obtained from absorbance at 280 nm, while dashed lines depict absorbance at 410 nm indicating [2Fe-2S] cluster binding (0.1–0.4 mg protein loaded). The elution positions and sizes of the molecular mass standards are shown above the chromatograms.

Figure 3

CD-monitored titration of [2Fe-2S]-GRX domain with Fra2(C29A), Fra2(H66A), and Fra2(C29A,H66A). [2Fe-2S]-GRX domain homodimer was titrated with 1- to 15-fold equivalents of apo Fra2(C29A) (A), 1- to 20-fold equivalents of apo Fra2(H66A) (B) or 1- to 5-fold equivalents of apo Fra2(H66A, C29A) (C). D, comparison of the CD spectra of [2Fe-2S]-GRX domain, [2Fe-2S]-GRX domain + WT Fra2, [2Fe-2S]-GRX domain + Fra2(C29A), [2Fe-2S]-GRX domain + Fra2(C66A), and [2Fe-2S]-GRX domain + Fra2(C29A/H66A). Δε values are based on the [2Fe-2S] cluster concentration (47 μM for A and B, and 100 μM for C). Arrows at selected wavelengths indicate the direction of change in peak intensity with increasing Fra2 concentrations. In A–C, thick black lines are the CD spectra of [2Fe-2S]-GRX domain alone and blue, green, and purple lines are the final titration mixtures. E, % CD intensity changes (between 408 and 458 nm) from spectra in A–C (Fra2 mutants) and Figure 1B (WT Fra2) are plotted as a function of the Fra2 concentration. The points were fit to Equation 8 to calculate the K_eq for each Fe-S exchange reaction (Table 1).

Figure 4

Cys29 and His66 amino acid residues of Fra2 are required to allow cell growth under low-iron conditions. Cells expressing fra2⁺-Myc₁₃, fra2C29A-Myc₁₃, fra2H66A-Myc₁₃, and fra2C29A-H66A-Myc₁₃ alleles were spotted onto YES medium that was left untreated or supplemented with Dip (150 μM). As controls, WT (fra2⁺) and fra2Δ strains were assayed under the same conditions. Once spotted on the untreated and iron-starved media, the strains were incubated for 4 days at 30 °C, and photographed.

Figure 5

Effects of the expression of fra2⁺-Myc₁₃, fra2C29A-Myc₁₃, fra2H66A-Myc₁₃, and fra2C29A-H66A-Myc₁₃alleles on the transcriptional response of frp1⁺to iron starvation. Representative expression profile of the frp1⁺ transcript in cells expressing the WT fra2⁺-Myc₁₃ or its mutant derivatives that were left untreated (−) or were incubated in the presence of Dip (250 μM) or FeCl₃ (Fe, 100 μM) for 90 min. Total RNA was prepared from culture aliquots, and steady-state mRNA levels of frp1⁺ and act1⁺ were analyzed by RT-qPCR assays. Graphic representation of quantification of three independent RT-qPCR assays. Error bars indicate the standard deviation (±SD; error bars). The asterisks correspond to p ˂ 0.01 (∗∗) and p < 0.0001 (∗∗∗∗) (two-way ANOVA with Tukey’s multiple comparisons test against the indicated strain grown under low-iron conditions), whereas ns stands for not significant.

Figure 6

CD-monitored titration of [2Fe-2S],Zn-Fep1-DBD with apo-Grx4/GRX domain and apo-Fra2. [2Fe-2S],Zn-Fep1-DBD was titrated with 0.25 to 12 mol equivalents of apo-Grx4 (A), apo-GRX domain (B), apo-Grx4-Fra2 (C), or apo-GRX domain-Fra2 (D). The arrows at selected wavelengths indicate the direction of CD intensity change with increasing apo-acceptor protein. Δε values are based on the [2Fe-2S] cluster concentration (50 μM). In A–D, thick black lines are the CD spectra of [2Fe-2S],Zn-Fep1-DBD alone, blue lines are the final titration mixtures with Grx4 or GRX domain, and purple lines are the final titration mixtures with Grx4-Fra2 or GRX domain-Fra2. E and F, % CD intensity changes (between 408 and 458 nm for A and B, and between 362 and 436 nm for C and D) from spectra in A and C (apo-Grx4 ± apo-Fra2) and B and D (apo-GRX domain ± apo-Fra2) are plotted as a function of the concentration of apo acceptor (dimer). The points were fit to Equation 8 to calculate the K_eq for each Fe-S exchange reaction (Table 1).

Figure 7

Model for Grx4-Fra2 dependent regulation of Fep1 repressor activity. See Discussion for details.