Overlapping binding sites of the frataxin homologue assembly factor and the heat shock protein 70 transfer factor on the Isu iron-sulfur cluster scaffold protein

Abstract

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.

Keywords: Frataxin; Iron-Sulfur Protein; Mitochondria; Molecular Chaperone; Protein-Protein Interaction; Saccharomyces cerevisiae.

FIGURE 1.

Stable Yfh1 binding requires the presence of both Nfs1(Isd11) and Isu1. GST-Yfh1 pulldown (A–D). Glutathione resin was used to pull down GST-Yfh1 (or GST) and any associated proteins. Pelleted proteins were separated by SDS-PAGE and stained. A, 2.5 μm GST-Yfh1 or GST was mixed with 15 μm Nfs1(Isd11) and/or Isu1 as indicated (±). B, 2.5 μm GST-Yfh1 was mixed with 15 μm Isu1 and the indicated concentrations of Nfs1(Isd11) (left panel) or with 15 μm Nfs1(Isd11) and the indicated concentrations of Isu1 (right panel). Densitometry values plotted as relative units (r.u.) with maximum binding Nfs1 (left) and Isu1 (right) were set at 1. C, 2.5 μm GST-Yfh1 or GST was mixed with 15 μm Nfs1(Isd11) and WT Isu1 or Isu1^{L63A/V72A/F94A} variant (LVF). Broken line indicates where the gel was cropped. D, 2.5 μm GST-Yfh1 was mixed with 15 μm Nfs1(Isd11) and indicated concentrations of Isu1 (WT) or Isu1^{L63A/V72A/F94A} variant (LVF). Samples were quantified as described in B. E, cysteine desulfurase activity of Nfs1(Isd11) measured in the absence and presence of the indicated proteins. Activity of Nfs1(Isd11) alone was set to 1. Bars represent average value for three independent measurements, with error bars indicating the range of the measurements.

FIGURE 2.

Structural model of bacterial FeS cluster assembly complex. PDB file of bacterial FeS assembly complex (13) was kindly provided by Annalisa Pastore (King's College, London). Residues predicted to be critical for frataxin interaction with the FeS cluster scaffold and cysteine desulferase are listed for bacterial proteins (CyaY, IscU, and IscS) and their yeast orthologs (Yfh1, Isu1, and Nfs1, respectively), as indicated.

FIGURE 3.

Replacement of Yfh1 Trp¹³¹ by alanine results in defective interaction with Isu1 of the Isu1-Nfs1(Isd11) complex. A, 2.5 μm GST-Yfh1 (WT) or GST-Yfh1^W131A (W131A) was mixed with 15 μm Nfs1(Isd11) and at the indicated concentrations of Isu1. Glutathione resin was added to pull down the GST-Yfh1 and associated proteins, which were separated by SDS-PAGE and stained. Densitometry values plotted as relative units (r.u.) with maximum binding set at 1. B, cysteine desulfurase enzymatic activity of Nfs1(Isd11) was measured in the absence and presence of the indicated proteins. Desulfurase activity of Nfs1(Isd11) alone was set to 1. Bars represent the average value for three independent measurements, with error bars indicating the range of measurements. C, 10-fold serial dilutions of yfh1-Δ cells harboring plasmid-borne copy of YFH1 (WT), yfh1^W131A (W131A), or plasmid lacking an insert (−), as indicated, were plated on glucose-rich medium and incubated at 30 °C for 3 days. D, equivalent amounts of cells described in C were solubilized as described under “Experimental Procedures” and subjected to immunoblot analysis using antibodies specific for Yfh1 and, as a control, Mge1. Strains (C) and samples (D) irrelevant to this study were removed from the image as indicated by a dotted line.

FIGURE 4.

PVK motif of Isu1 is critical for stable Yfh1 interaction with the Isu1-Nfs1(Isd11) complex. A, 2.5 μm GST-Yfh1 and 15 μm Nfs1(Isd11) was mixed with the indicated concentrations of Isu1 (WT) or indicated variant having alanine substitutions in place of Pro¹³⁴, Val¹³⁵, and/or Lys¹³⁶, as indicated. Glutathione resin was added to pull down the GST-Yfh1 and associated proteins, which were separated by SDS-PAGE and stained. Densitometry values plotted as relative units (r.u.) with maximum binding of WT Isu1 protein set at 1. B, 2.5 μm Isu1-GST (WT) or the indicated replacement variant was mixed with 15 μm Nfs1(Isd11). Glutathione resin was added to pull down the GST-Yfh1 and associated proteins, which were separated by SDS-PAGE and stained. Binding of WT was set at 1. Bars represent average value for three independent measurements, with error bars indicating the range of the measurements. C, cysteine desulfurase activity of Nfs1(Isd11) was measured in the absence and presence of the indicated proteins. Activity of Nfs1(Isd11) alone was set to 1. Bars represent average value for three independent measurements, with error bars indicating the range of the measurements. D, 2.5 μm Isu1-GST (WT) or Isu1^{P134A/V135A/K136A}-GST variant (PVK) or GST were incubated in the presence (+) or absence (−) of 4 μm Ssq1 and/or 0.25 μm Jac1. Glutathione resin was added to pull down GST and associated proteins, which were separated by SDS-PAGE and stained. E, 2.5 μm Isu1-GST, WT, or the indicated variants, were mixed with 4 μm Ssq1 and varying concentrations of Jac1. Glutathione resin was added to pull down GST and associated proteins, which were separated by SDS-PAGE and stained. Densitometry values plotted as relative units (r.u.) with maximum binding of Ssq1 set at 1.

FIGURE 5.

Replacement of residues Asp⁸⁶ and Glu⁸⁹ of Yfh1 results in reduced interaction with Nfs1 of the Isu1-Nfs1(Isd11) complex. A, 2.5 μm GST-Yfh1, WT, or variants having Asp⁸⁶ and Glu⁸⁹ replaced by Ala or Lys (DE/AA and DE/KK, respectively) was mixed with 15 μm Isu1 and the indicated concentrations of Nfs1(Isd11). Glutathione resin was added to pull down the GST-Yfh1 and associated proteins, which were then separated by SDS-PAGE and stained. Densitometry values plotted as relative units (r.u.) with maximum binding of Nfs1 set at 1. Bars represent average values for three independent measurements, with error bars indicating the range of the measurements. B, cysteine desulfurase activity of Nfs1(Isd11) was measured in the absence and presence of the indicated proteins. Desulfurase activity of Nfs1(Isd11) alone was set to 1. Bars represent the average value for three independent measurements, with error bars indicating the range of measurements. C, 10-fold serial dilution of yfh1-Δ cells harboring plasmid-borne copy of YFH1 (WT), yfh1^D86K/E89K (DE/KK) or plasmid lacking an insert (−) were plated on glucose-rich medium and incubated at 30 °C for 3 days. D, equivalent amounts of cells described in C were solubilized and subjected to immunoblot analysis using antibodies specific for Yfh1 and, as a control, Mge1. Strains (C) and lanes (D) irrelevant to this study were removed from the images as indicated by dotted line.

FIGURE 6.

Replacement of residues Arg³¹³, Arg³¹⁶, and Arg³¹⁸ of Nfs1 results in reduced binding of Yfh1 to the Isu1-Nfs1(Isd11) complex. A, 2.5 μm GST-Yfh1 was mixed with 15 μm Isu1 and the indicated concentrations of Nfs1(Isd11); wild-type (WT), Nfs1^{R313A/R316A/R318A} (RRR/AAA), or Nfs1^{R313E/R316E/R318E} (RRR/EEE). Glutathione resin was added to pull down GST-Yfh1 and associated proteins, which were separated by SDS-PAGE and stained. Densitometry values plotted as relative units (r.u.) with maximum binding of WT Nfs1 protein set at 1. B, 2.5 μm Isu1-GST was mixed with 15 μm Nfs1(Isd11) (WT) or the indicated replacement variants. Samples were treated and quantitated as in A. C, enzymatic activity of cysteine desulfurase Nfs1(Isd11) (WT) and the indicated replacement variants were measured in the absence and presence of other proteins, as indicated. Desulfurase activity of WT Nfs1(Isd11) alone was set to 1. B and C, bars represent average values for three independent measurements, with presented error bars indicating the range of the measurements. D, nfs1-Δ cells harboring an URA3-marked plasmid containing the WT copy of NFS1 and a second plasmid harboring either NFS1 (WT) or nfs1^RRR/AAA or nfs1^RRR/EEE or plasmid lacking an insert (−) were plated on glucose-minimal medium containing 5-fluoroorotic acid, which selects for cells having lost the plasmid containing the URA3 marker. The plate was incubated at 30 °C for 3 days. E, lysates of GAL-NFS1 cells transformed with plasmids having no insert (−) or harboring either a WT copy of NFS1 (WT), nfs1^RRR/AAA, or nfs1^RRR/EEE, as indicated, under the control of the native NFS1 promoter were prepared 24 h after transfer from galactose- to glucose-based medium and separated by SDS-PAGE. Immunoblots were probed with antibodies specific to Nfs1 and Mge1 (left) or porin (right), as a loading control. Left, whole cell lysates. Right, mitochondrial lysates.

FIGURE 7.

Residues involved in Yfh1 interaction with the Isu1-Nfs1(Isd11) complex are evolutionary conserved. Residues occupying positions homologous to those involved in Yfh1-Isu1 and Yfh1-Nfs1 interactions with Isu1-Nfs1(Isd11) complex are indicated for orthologous proteins from proteobacteria (386 species), α-proteobacteria (36 species), and eukaryotes (199 species). The percentage of species having given residues is indicated.

FIGURE 8.

Ordered transition from FeS cluster assembly to transfer. A, homology model of Isu1 (39) with highlighted residues involved in Yfh1-Ssq1 (red) and Nfs1-Jac1 (yellow) interaction with Isu1, as part of the FeS cluster assembly complex. B, Yfh1 binds the pre-formed Isu1-Nfs1(Isd11) complex (I) facilitating FeS cluster synthesis. Jac1 displaces holo-Isu1 from the assembly complex (II) to form the holo-Isu1-Jac1 complex. Ssq1 binds the PVK motif of holo-Isu1 in complex with Jac1 (III). J-domain (J) of Jac1 stimulates the ATPase activity of Ssq1 facilitating FeS cluster transfer (IV) to the recipient apoprotein.