Binding of the chaperone Jac1 protein and cysteine desulfurase Nfs1 to the iron-sulfur cluster scaffold Isu protein is mutually exclusive

Abstract

Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch critical for interaction with Isu. The results of molecular modeling of the Isu1-Jac1 interaction, which was guided by these experimental data and structural/biophysical information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was experimentally validated by in vitro binding assays. In addition, Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary analysis revealed that residues involved in these interactions are conserved and that they are critical residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.

Keywords: ISC System; Iron-Sulfur Protein; J-protein; Mitochondria; Molecular Chaperone; Scaffold Proteins; Yeast.

FIGURE 1.

Replacement of the Leu⁶³, Val⁷², and Phe⁹⁴ residues of Isu1 results in defective Jac1 binding and the inability to support cell growth. A, model of the Jac1-Isu1 complex (center panel) on the basis of in silico docking of the Jac1 protein crystal structure (PDB code 3UO3, left panel) and homology model of the Isu1 structure (right panel). Residues of Jac1 and Isu1 implicated in their interaction are highlighted. B, Isu1-GST-Jac1 pull-down. 2.5 μm Isu-GST was mixed with the indicated concentrations of Jac1: WT Jac1 with WT Isu1-GST (WT), Isu1^{L63,V72,F94/AAA} (LVF/AAA), or Isu1^{L63,V72,F94/SSS} (LVF/SSS) and WT Isu1-GST with WT Jac1 (WT), Jac1^Y163/A (Y/A), or Jac1^{L105,L109,Y163/AAA} (LLY/AAA). Glutathione resin was added to pull down the complex. Isu1-GST and Jac1 were separated by SDS-PAGE, visualized by staining, and quantitated by densitometry. Values were plotted in GraphPad Prism using 1:1 binding hyperbola to fit data and plotted as relative units (r.u.), with maximum binding of WT protein given a value of 1. C, (left panel) jac1-Δ cells harboring a plasmid-borne copy of WT JAC1 (on an URA3-based plasmid and a second plasmid harboring WT JAC1 (WT), jac1^{L105,L109,Y163/AAA} (LLY/AAA), or jac1^Y163/A (Y/A). Right panel, isu-Δ cells harboring a plasmid-borne copy of WT ISU1 (URA3-marked) and a second plasmid harboring WT ISU1(WT), isu1^{L63,V72,F94/AAA} (LVF/AAA), or isu1^{L63,V72,F94/SSS} (LVF/SSS). Strains were plated on glucose-minimal medium containing 5-fluorootic acid, which selects for cells having lost the plasmid containing the URA3 marker, and incubated at 30 °C for 3 days. D, lysates of GAL-ISU1:isu2-Δ cells transformed with either a plasmid lacking an insert (-) or a WT copy of ISU1, isu1^LVF/AAA, isu1^LFV/SSS, under the control of the native ISU1 promoter, were prepared 17 h after transfer from galactose- to glucose-containing medium and separated by electrophoresis. Immunoblots were probed with antibodies specific to Isu1 and actin, a loading control.

FIGURE 2.

Replacement of residues Leu⁶³, Val⁷², and Phe⁹⁴ of Isu1 results in defective interaction with Nfs1(Isd11) in vitro. A, Nfs1(Isd11), at the indicated concentrations, was mixed with 2.5 μm Isu1-GST (left) or incubated in the absence of GST fusion (right). Glutathione resin was added to pull down the complex. Proteins were separated by electrophoresis and visualized by staining. B, top panel, schematic of variants tested. Bottom panel, Isu1-GST (2.5 μm) WT and variants, as indicated, were treated as described in A. Results were quantitated by densitometry, and obtained values were plotted in GraphPad Prism using a 1:1 binding hyperbola to fit data and plotted as relative units (r.u.), with WT Isu1 given a value of 1.

FIGURE 3.

Replacement of residues Leu⁴⁷⁹ and Met⁴⁸² of Nfs1 results in reduced interaction with Isu1 and the inability to support cell growth. A, homology model of the Nfs1-Isu1 complex on the basis of the crystal structure of the complex of bacterial orthologous proteins (IscS-IscU, PDB code 3LVL). Residues implicated in Nfs1-Isu1 interaction pertinent to this work are highlighted. B, Isu1-GST (2.5 μm), WT or variants, as indicated, were mixed with Nfs1(Isd11) at the indicated concentrations to allow complex formation. Glutathione resin was added to pull down the complex. Proteins were separated by electrophoresis, visualized by staining, and quantitated by densitometry. Values were plotted in GraphPad Prism using a 1:1 binding hyperbola to fit data and plotted as relative units (r.u.), with maximal binding of WT Nfs1 given a value of 1. L479, Nfs1^L479/A; M482, Nfs1^M482/A; LM, Nfs1^L479,M482/AA. C, top panel, nfs1-Δ cells harboring an URA3-marked plasmid containing the WT NFS1 (WT) and a second plasmid harboring either WT NFS1 or nfs1^L479,M482/AA (LM) were plated on glucose-minimal medium containing 5-fluorootic acid, which selects for cells having lost the plasmid containing the URA3 marker. The plate was incubated at 30 °C for 3 days. Bottom panel, lysates of GAL-NFS1 cells transformed with plasmids having no insert (-) or harboring either a WT copy of NFS1 (WT) or nfs1^L479,M482/AA under the control of the native NFS1 promoter were prepared 22 h after transfer from galactose- to glucose-based medium and separated by SDS-PAGE. Immunoblots were probed with antibodies specific to Nfs1 and porin, a loading control. D, cysteine desulfurase activity of purified WT and the Leu⁴⁷⁹Met⁴⁸²/AA variant (LM) Nfs1(Isd11) was measured. E, aconitase activity (Aco) and succinate dehydrogenase activity were measured in lysates of mitochondria isolated from GAL-ISU1 isu2-Δ cells harboring plasmid-borne copies of ISU1 (WT), Isu1^{L63V72F94/AAA} isu1^LVF/AAA, or vector without insert (-), grown for 17 h after transfer from galactose- to glucose-containing medium. As a standard, the enzymatic activity of non-Fe/S cluster-containing protein malate dehydrogenase (MDH) was measured. The ratio of activities of aconitase or SDH and malate dehydrogenase was calculated and expressed as a percentage of the WT control. Bars represent average values for three measurements, with presented error bars as S.D. F, aconitase activity and SDH activity were measured in lysates of mitochondria isolated from GAL-NFS1 cells harboring plasmid-borne copies of WT NFS1, nfs1LM/AA, or vector without insert, as indicated, grown for 40 h in glucose-containing medium. Enzymatic activities were measured and plotted as described in E.

FIGURE 4.

Nfs1 and Jac1 compete with each other for binding to Isu1. Isu1-GST (2.5 μm) was mixed with either 5 μm Nfs1(Isd11) or 5 μm Jac1 to allow complex formation. Incubation was continued after addition of the indicated concentration of the competitor (Jac1, after preincubation with Nfs1(Isd11) (A) and Nfs1(Isd11) after preincubation with Jac1 (B)). Glutathione resin was added to pull down Isu1-GST complexes. Proteins were separated by SDS-PAGE, visualized by Coomassie Blue staining, and quantitated by densitometry. Data were plotted as relative units (r.u.) with binding in the absence of a competitor given a value of 1.

FIGURE 5.

Residues involved in Nfs1/Jac1 interaction with Isu1 are evolutionary conserved. Residues occupying positions homologous to those involved in Nfs1/Jac1 interaction with Isu1 are indicated for orthologous proteins from proteobacteria (358 species), α-proteobacteria (32 species), fungi (62 species), and other eukaryotic (22 species (see supplemental Table S1 for the list of species). The percentage of species having given residues is indicated.

FIGURE 6.

Jac1 involvement in the transition from Fe/S assembly to transfer. A, when an Fe/S cluster is synthesized on the Isu1 scaffold via action of the assembly complex (left), Jac1 displaces Nfs1 (center) and Jac1 targets Isu1-Fe/S for mtHsp70 binding, facilitating cluster transfer to recipient apoprotein (right). B, homology model of Isu1 with highlighted residues involved in both Nfs1 and Jac1 binding (orange), Jac1 binding only (brown), and Nfs1 binding only (yellow).