Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

Abstract

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.

Fig. 1. Stable binding of an Fe/S cluster to Isu1p. (A) Yeast cells overproducing (↑) wild-type Isu1p or the site-directed mutant Isu1p^C96S from the plasmid p426GPD were grown in ‘iron-poor’ minimal medium with glucose for 16 h. Control cells (–) contained the empty plasmid. Cells were radiolabelled with ⁵⁵Fe for 2 h and cell lysates were prepared (Kispal et al., 1999). Isu1p was immunoprecipitated with anti-Isu1p antibodies under aerobic or anaerobic (–O₂) conditions and the amount of radioactivity coprecipitated with the immuno-beads was quantitated by liquid scintillation counting. A control immuno precipitation was performed with preimmune serum (PIS). The inset shows an immunostaining of Isu1p in extracts from wild-type cells (–) or cells overproducing Isu1p or Isu1p^C96S (↑). Error bars indicate the standard deviation of the measurements. (B) Isu1p and Nfs1p were purified after overproduction in Escherichia coli. Isu1p (10 µg) and Nfs1p (5 µg) were incubated with ⁵⁵Fe in the presence or absence of cysteine (Cys) for 150 min under anaerobic conditions developed for in vitro reconstitution of mitochondrial Fe/S protein biogenesis (Mühlenhoff et al., 2002a). A labelling reaction lacking Isu1p (–Isu1p) served as control. Reactions were terminated with EDTA, Isu1p was immunoprecipitated and the amounts of co-immunoprecipitated ⁵⁵Fe were determined by scintillation counting.

Fig. 2. Assembly of an Fe/S cluster on Isu1p in vivo requires the cysteine desulfurase Nfs1p. Wild-type (WT) and Gal-NFS1 cells overproducing (↑) (A) Isu1p or (B) an HA-tagged version of Yah1p (Yah1p-HA) were incubated in iron-poor medium supplemented with galactose (SGal) or glucose (SD) in order to downregulate the expression of NFS1 in Gal-NFS1 cells. Cells were radiolabelled with ⁵⁵Fe, Isu1p and Yah1p-HA were immunoprecipitated from the cell lysates and the amount of co-immunoprecipitated ⁵⁵Fe was quantified by scintillation counting. As a control, wild-type cells that did not overproduce the two proteins were analysed. The insets show the immunostaining of the indicated mitochondrial proteins in extracts derived from Gal-NFS1 cells grown in the different minimal media.

Fig. 3. Fe/S cluster assembly on Isu1p depends on the ferredoxin Yah1p. (A) Gal-YAH1 cells overproducing (↑) Isu1p and (B) Gal-ISU1/Δisu2 cells overproducing HA-tagged Yah1p were cultivated in iron-poor SGal or SD minimal media in order to allow or repress, respectively, synthesis of Yah1p and Isu1p. In parallel, wild-type (WT) cells were analysed. After radiolabelling with ⁵⁵Fe, Isu1p or Yah1p-HA were immunoprecipitated from cell lysates and the amounts of co-immunoprecipitated ⁵⁵Fe were determined. The insets show immunostaining of various mitochondrial proteins in extracts derived from the two mutant cells after growth in the indicated media.

Fig. 4. Depletion of frataxin (Yfh1p) results in decreased Fe/S cluster formation on Isu1p. Gal-YFH1 cells overproducing (↑) Isu1p and Yah1p-HA or wild-type (WT) cells were grown under conditions permissive (SGal) and repressive (SD) for synthesis of Yfh1p. After radiolabelling with ⁵⁵Fe and cell lysis, immunoprecipitations were carried out using specific antisera and the amounts of co-immuno precipitated radioactivity were quantified. The insets show immuno staining of various proteins in extracts derived from Gal-YFH1 cells.

Fig. 5. The Hsp70 chaperone Ssq1p is involved in the de novo maturation of mitochondrial Fe/S proteins, but not required for Fe/S cluster assembly on Isu1p in vivo. Iron-starved wild-type (WT), Δssq1 and Gal-SSQ1 cells overproducing (↑) (A) Isu1p, (B) Yah1p-HA and (C) Bio2p (grown as in Figure 2) were radiolabelled with ⁵⁵Fe. Immunoprecipitation was carried out using specific antisera against these proteins and the amounts of co- immunoprecipitated radioactivity were determined by liquid scintillation counting. (D) Immunostaining of various mitochondrial proteins in extracts from Gal-SSQ1 cells grown in galactose-containing (SGal) or glucose-containing (SD) minimal media.

Fig. 6. Increased accumulation of an Fe/S cluster on Isu1p upon depletion of Jac1p in vivo. Gal-JAC1 cells overproducing (↑) (A) Isu1p, (B) Yah1p-HA and (C) Bio2p (grown as in Figure 2) were labelled with radioactive ⁵⁵Fe in vivo, immunoprecipitations were carried out using specific antibodies against the three overproduced proteins and the amounts of co-immunoprecipitated radioactivity were quantified. As a control, wild-type (WT) cells that did not overproduce these proteins were analysed. (D) Immunostaining of various mitochondrial proteins in extracts from Gal-JAC1 cells grown in the presence of galactose (SGal) or glucose (SD).

Fig. 7. Depletion of the mitochondrial glutaredoxin Grx5p leads to accumulation of Fe/S clusters on Isu1p. Gal-GRX5 cells were transformed with plasmids for overproduction (↑) of (A) Isu1p, (B) Yah1p-HA and (C) Bio2p. Analyses for ⁵⁵Fe radiolabelling of these proteins by immunoprecipitation and the evaluation of the data was carried out as described in Figure 5. (D) Immunostaining of the indicated proteins after growth in the media used.

Fig. 8. Working model for the biogenesis of Fe/S proteins in mitochondria. The essential protein pair Isu1p and Isu2p plays a crucial role in biogenesis by serving as a scaffold for de novo assembly of an Fe/S cluster. As defined here, this reaction crucially involves the cysteine desulfurase Nfs1p, the electron transport chain Yah1p/Arh1p and Yfh1p (frataxin) providing an unknown function. The chaperones Ssq1p/Jac1p and the glutaredoxin Grx5p are not needed for Fe/S cluster synthesis on Isu1/2p; rather, they may function in the dislocation of the Fe/S cluster from Isu1/2p and/or its insertion into the apoproteins. The requirement of reduced iron, NADH, ATP and cysteine has been documented by in vitro reconstitution of the Fe/S protein assembly reaction (Mühlenhoff et al., 2002a). Iron import into mitochondria by an unknown transporter is driven by a proton motive force (pmf) (Lange et al., 1999).