A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery

Abstract

Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximity-dependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3·BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, whereas Glrx3 homodimer formation did not. Cellular Glrx3·BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly expandable pool of Fe-S clusters. Fe-S coordination by Glrx3·BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3·BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apoproteins in human cells.

Keywords: BolA2; Ciapin1; Glrx3; Ndor1; biotin; iron; iron-sulfur protein; metal homeostasis; metal ion-protein interaction; metalloprotein.

FIGURE 1.

Glrx3·BolA2 forms an Fe-S-dependent complex in human cells. A, physical interaction between Glrx3 and BolA2 in cells. HEK293 cells containing an empty vector (EV) or inducible Glrx3-FLAG (Glrx3-F) or BolA2-FLAG (BolA2-F) were treated overnight with doxycycline. Cells were harvested and lysed, and anti-FLAG immunoprecipitation (IP) was performed. Whole cell extracts (WCE) and immune complexes (IP: FLAG) were analyzed by immunoblotting using antibodies against Glrx3 and BolA2. B, iron-dependent formation of the Glrx3·BolA2 complex. BolA2-FLAG cells were treated with doxycycline and 50 μm Dfo or 100 μm FeCl₃ for 16 h. WCE and anti-FLAG immune complexes were analyzed by immunoblot. C, requirement of Nfs1 for Glrx3·BolA2 complex formation. BolA2-FLAG cells were treated with siRNAs against Nfs1 or a non-targeting control (Con), induced with doxycycline, and analyzed by IP and immunoblotting. Ratio of co-precipitated Glrx3 to IP BolA2-FLAG, normalized to control, is shown at right. n = 3, error bars represent S.E. D, depletion of Nfs1 using siRNA. Immunoblotting analysis of whole cell extracts from BolA2-FLAG cells treated with control siRNA or siRNA against Nfs1 for 4 days. E, failure of iron to restore Glrx3·BolA2 complex formation in Nfs1-depleted cells. BolA2-FLAG cells were depleted of Nfs1 as in A, then treated with or without 25 μm FeCl₃ overnight. Immunoblotting analysis of whole cell extracts and anti-FLAG immune complexes is shown.

FIGURE 2.

Fe-S cluster binding residues of Glrx3 are required for stabilizing the Glrx3·BolA2 interaction. A, schematic of BirA*-FLAG-Glrx3. Deletion and amino acid substitutions of BrF-Glrx3 mutants are shown. B, impaired interaction between BolA2 and Glrx3 Fe-S-binding mutants. Anti-FLAG immune complexes were isolated from cells expressing EV or the indicated allele of BrF-Glrx3. WCE (B) and immune complexes (C) were analyzed by immunoblot. Ratio of co-precipitated BolA2 to BrF-Glrx3 in IPs, normalized to WT, is shown at right. n = 3, error bars represent S.E.

FIGURE 3.

Glrx3·BolA2 complex coordinates a rapidly expandable pool of Fe-S clusters. A, iron-dependent increases in Glrx3·BolA2 complexes. BolA2-FLAG cells were induced and treated with the indicated concentration of Dfo or FeCl₃ for 16 h. WCE and anti-FLAG immune complexes were prepared and analyzed by immunoblotting using antibodies against Glrx3 and FLAG. B, quantitation of IPs in A. Ratio of co-precipitated Glrx3 to BolA2-FLAG in IP was normalized to 50 μm Dfo. C, rapid reformation of Glrx3·BolA2 complexes from apo-BolA2 after cellular iron repletion. Induced BolA2-FLAG cells were treated overnight with 50 μm Dfo to promote formation of apo-BolA2-FLAG. Dfo medium was replaced with 50 μm FeCl₃ medium for the indicated times. Whole cell extracts, anti-FLAG IPs, and post-IP supernatants were prepared and analyzed by immunoblotting against Glrx3 and FLAG. Quantitative comparisons of Post-IP Supernatant and WCE were made from samples loaded in parallel on same immunoblot. D, quantitation of IPs in C. E, rapid reformation of Glrx3·BolA2 complexes from apo-FLAG-Glrx3. FLAG-Glrx3 cells were grown and analyzed as in C, above, using antibodies against BolA2 and FLAG. In all experiments n = 3, error bars represent S.E.

FIGURE 4.

Glrx3 homodimers are detectable in live cells and more abundant in iron-limited cells. A, detection of Glrx3 homodimers in human cells using BrF-Glrx3. Lysates from biotin-supplemented cells stably expressing inducible BirA*-FLAG (BrF) or BirA*-FLAG-Glrx3 (BrF-Glrx3) were subjected to biotin capture using streptavidin-agarose. Captured biotinylated proteins were eluted and probed for endogenous Glrx3 using immunoblotting analysis (arrowhead). Total biotinylated protein was detected using IR dye-conjugated streptavidin. B and C, increased Glrx3 homodimer formation in iron-deficient cells. Cells expressing BrF-Glrx3 were made iron-deficient or -replete by overnight treatment with 25 μm Dfo or 100 μm FeCl₃, respectively. Following biotin supplementation for the indicated times, cells were harvested and lysed, and the resulting whole cell extracts were subjected to biotin capture and immunoblot analyses as described in A. D, similar levels of biotinylated proteins in WCE from iron- and Dfo-treated cells. Induced BrF-Glrx3 cells were treated as in B and C for the indicated times. Immunoblotting analysis of total biotinylated protein is shown.

FIGURE 5.

Glrx3 homodimers are not stabilized by Fe-S clusters. A, BolA2 deficiency does not increase Glrx3 homodimerization. Cells expressing BrF-Glrx3 were treated with siRNA against BolA2 and analyzed for Glrx3 homodimer formation by capture of biotinylated Glrx3 (arrow). Quantitation of captured endogenous Glrx3 is shown in middle. n = 4; error bars represent S.E. Immunoblotting analysis of total biotinylated proteins is shown at right. Con, control. n = 4; error bars represent S.E.; **, p < 0.002. B, lack of effect of hypoxia on Glrx3 homodimer formation. BrF-Glrx3 cells supplemented with biotin were grown under 19 or 5% O₂ for 20 h. Cells lysates were prepared, and biotinylated proteins were captured using streptavidin-agarose resin. Captured proteins were eluted and probed for Glrx3 and total biotin using immunoblotting analysis. C, lack of effect Glrx3 Fe-S cluster coordination on homodimerization. Biotinylated endogenous Glrx3 was captured from Dfo- and iron-treated cells stably expressing wild type (WT) or Fe-S binding mutant (AB^M) of BrF-Glrx3.

FIGURE 6.

Ciao1 binds Glrx3 but is not required for Glrx3·BolA2 cluster acquisition. A, binding of endogenous Ciao1 with Glrx3 but not BolA2. EV, Glrx3-FLAG (Glrx3-F), or BolA2-FLAG (BolA2-F) cells were treated with 50 μm Dfo or 100 μm FeCl₃. WCE were prepared and analyzed by anti-FLAG IP and immunoblotting analysis using antibodies against Ciao1and FLAG. B, no effect of Ciao1 depletion on Glrx3·BolA2 interaction. Cells expressing BolA2-FLAG were treated with control (Con) or Ciao1 siRNA. WCE and anti-FLAG immune complexes were analyzed by immunoblot.

FIGURE 7.

Ciapin1 primarily interacts with Glrx3 via its Fe-S clusters and Trx domain. A, co-precipitation of endogenous Ciapin1 with Glrx3 or BolA2. Glrx3-FLAG (Glrx3-F) and BolA2-FLAG (BolA2-F) cells were treated with 50 μm Dfo or 100 μm FeCl₃, followed by IP. Immunoblotting analysis of Ciapin1 (Cpn) from WCE, post-IP supernatant (post-IP Sup), and anti-FLAG immune complexes was performed. B, interaction of endogenous Glrx3 and BolA2 with Ciapin1-FLAG. Cells containing an EV or expressing inducible Ciapin1-FLAG (Cpn-F) were analyzed by anti-FLAG IP and immunoblotting using antibodies against Glrx3, BolA2, and Cpn. C, requirement of Glrx3 for interaction of BolA2 with Ciapin1. Cells expressing Ciapin1-FLAG were treated with control, Glrx3, or BolA2 siRNA. Whole cell extracts were subjected to IP and immunoblotting analysis as in C. Dox, doxycycline. D, requirement of Fe-S cluster and Trx domain of Glrx3 in interaction with Ciapin1. Lysates from cells expressing BrF alone or BrF-Glrx3 wild type (WT), thioredoxin deletion mutant (ΔTrx), or Fe-S cluster mutant (AB^M) alleles were subjected to anti-FLAG IP. Whole cell extracts and anti-FLAG immune complexes were analyzed by immunoblotting using antibodies against FLAG and Ciapin1. E, co-precipitation of Ndor1 with Ciapin1, Glrx3, and BolA2. Immunoblotting analysis for Ndor1 from anti-FLAGs IPs of Ciapin1-FLAG, Glrxe-FLAG, and BolA2-FLAG cells. F, co-precipitation of Ndor1 with Ciapin1 in cells lacking Glrx3 or BolA2. Cells expressing Ciapin1-FLAG were treated as in C, above, and IPs subjected to immunoblotting analysis for Ndor1.

FIGURE 8.

Glrx3 and BolA2 are required for stabilization of Ciapin1 protein. A, loss of Ciapin1 protein in cells lacking Glrx3 or BolA2. Immunoblotting analysis of cells expressing inducible Ciapin1-FLAG (Cpn-F) following two sequential treatments with siRNA against Glrx3 or BolA2. Quantitation at right. n = 4, ***, p < 0.002. Con, control; dox, doxycycline. B, decrease in endogenous Ciapin1 after depletion of Glrx3 or BolA2. HEK293T cells were treated with the indicated siRNA for 4 days. Cell lysates were then prepared and subjected to immunoblotting analysis using antibodies against Ciapin1 (Cpn) and Gapdh. C, no loss of Ciapin1 mRNA. RT-PCR analysis of Ciapin1 mRNA isolated from HEK293T cells treated with siRNA as described in B. Ciapin1 mRNA levels were normalized to Gapdh and expressed relative to control siRNA-treated cells. D, restoration of Ciapin1-FLAG levels by lysosomal inhibition. Ciapin1-FLAG cells received two treatments of control, Glrx3, or BolA2 siRNA. Cells were then induced with doxycycline in the presence or absence of 80 nm bafilomycin A for 12–16 h and then analyzed by immunoblotting. Quantitation at right. n = 3, error bars represent S.E., *, p < 0.05. E, decrease in Ciapin1-FLAG in Fe-S cluster-deficient cells. Ciapin1-FLAG (Cpn-F) cells were treated with control siRNA or siRNA against Nfs1 for 4 days. After Nfs1 depletion, Ciapin1-FLAG expression was induced with doxycycline. Whole cell extracts were analyzed by immunoblot using anti-FLAG and Gapdh antibodies.

FIGURE 9.

Glrx3 and BolA2 are required for iron incorporation into Ciapin1. A, isolation of ⁵⁵Fe-containing Ciapin1 complexes from cells. Ciapin1-FLAG cells were labeled with ⁵⁵FeCl₃ for 16 h. Whole cell extracts were analyzed by FLAG IP and scintillation counting. Retained ⁵⁵Fe is expressed relative to uninduced (−dox) cells. Dox, doxycycline. B, loss of ⁵⁵Fe in Ciapin1 complexes from cells lacking Glrx3 or BolA2. Ciapin1-FLAG cells treated with control (Con), Glrx3, or BolA2 siRNAs were given ⁵⁵FeCl₃ for 8 h. ⁵⁵Fe specifically bound to Cpn-F was measured as in A and normalized to total ⁵⁵Fe accumulation. Ciapin1-FLAG from whole cell extracts shown at right. n = 3, error bars represent S.E., **, p < 0.01. Immunoblot analyses of Ciapin1-FLAG, Glrx3, and BolA2 are shown at right panel. Immunoblots of Glrx3, BolA2, and Gapdh were made from same lysates shown in Fig. 8D. Gapdh blot also shown in Fig. 8D. C, transfer of Glrx3·BolA2 Fe-S clusters to Ciapin1. Ciapin1-FLAG cells treated with or without doxycycline were ⁵⁵Fe-labeled as described. Anti-FLAG IPs were performed in buffers and washes containing the indicated concentration of NaCl. Immune complexes were divided for ⁵⁵Fe quantitation by scintillation counting (left) and immunoblotting analysis of Glrx3 and Cpn1 with quantitation (right). n = 3, error bars represent S.E., **, p < 0.01.