Mitochondrial glutaredoxin Grx5 functions as a central hub for cellular iron-sulfur cluster assembly

Abstract

Iron-sulfur (Fe-S) protein biogenesis in eukaryotes is mediated by two different machineries-one in the mitochondria and another in the cytoplasm. Glutaredoxin 5 (Grx5) is a component of the mitochondrial iron-sulfur cluster machinery. Here, we define the roles of Grx5 in maintaining overall mitochondrial/cellular Fe-S protein biogenesis, utilizing mitochondria and cytoplasm isolated from Saccharomyces cerevisiae cells. We previously demonstrated that isolated wild-type (WT) mitochondria themselves can synthesize new Fe-S clusters, but isolated WT cytoplasm alone cannot do so unless it is mixed with WT mitochondria. WT mitochondria generate an intermediate, called (Fe-S)_int, that is exported to the cytoplasm and utilized for cytoplasmic Fe-S cluster assembly. We here show that mitochondria lacking endogenous Grx5 (Grx5↓) failed to synthesize Fe-S clusters for proteins within the organelle. Similarly, Grx5↓ mitochondria were unable to synthesize (Fe-S)_int, as judged by their inability to promote Fe-S cluster biosynthesis in WT cytoplasm. Most importantly, purified Grx5 precursor protein, imported into isolated Grx5↓ mitochondria, rescued these Fe-S cluster synthesis/trafficking defects. Notably, mitochondria lacking immediate downstream components of the mitochondrial iron-sulfur cluster machinery (Isa1 or Isa2) could synthesize [2Fe-2S] but not [4Fe-4S] clusters within the organelle. Isa1↓ (or Isa2↓) mitochondria could still support Fe-S cluster biosynthesis in WT cytoplasm. These results provide evidence for Grx5 serving as a central hub for Fe-S cluster intermediate trafficking within mitochondria and export to the cytoplasm. Grx5 is conserved from yeast to humans, and deficiency or mutation causes fatal human diseases. Data as presented here will be informative for human physiology.

Keywords: cytoplasm; export; iron; iron-sulfur protein; metal cofactor; mitochondria; sulfur; tRNA thiolation; yeast.

Figure 1

Correlating enzyme activity and [4Fe-4S] cluster loading of endogenous aconitase in isolated mitochondria. The native promoter of several genes (GRX5, ISA1, ISA2, and IBA57) was individually replaced with the GAL1 promoter in the genome (45), generating corresponding Gal strains (Table S1). The WT BY4741, Gal strains, and two deletion strains (Δnfu1 and Δbol3) were grown in raffinose plus dextrose medium (no galactose), and mitochondria were isolated. Under these conditions, the GAL1 promoter in Gal strains is turned off and expression of the corresponding protein is repressed (↓). A–F, left panels: isolated mitochondria (“mito”) were lysed, subjected to native PAGE, and analyzed for aconitase activity by an in-gel assay (44, 54). 1X = 50 μg of proteins. A–F, right panels: WT or mutant mitochondria (200 μg of proteins) were incubated with [³⁵S]cysteine (10 μCi), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), and ferrous ascorbate (10 μM) at 30 °C for 10 to 30 min. Reaction mixtures were diluted with isotonic buffer and centrifuged. The mitochondrial pellets thus obtained were analyzed by native PAGE, followed by autoradiography (40, 42). Grx5, glutaredoxin 5.

Figure 2

Effects of newly imported Grx5 or Isa1 into isolated mitochondria on endogenous aconitase [4Fe-4S] cluster loading.A, WT or Grx5-depleted (Grx5↓) mitochondria (“Mito”; 200 μg of proteins) were supplemented with [³⁵S]cysteine, nucleotides, iron, and as indicated, purified Grx5 precursor protein (pGrx5; 1X = 0.5 μg). Samples were incubated at 30 °C for 30 min, centrifuged, and mitochondrial pellets (“P”) were analyzed by native PAGE, followed by autoradiography. B, Grx5↓ mitochondria (200 μg of proteins) were supplemented with [³⁵S]cysteine, nucleotides, and iron, and then incubated at 30 °C for 10 to 30 min, with or without added pGrx5 protein (1 μg) as indicated. Samples were analyzed as in (A) above. C, mitochondria (WT or Grx5↓; 200 μg of proteins) were incubated on ice for 2 min, with or without valinomycin (5 μM). Assay mixtures were then supplemented with [³⁵S]cysteine, nucleotides, iron, and as indicated, pGrx5 protein (1 μg). After incubation at 30 °C for 30 min, samples were analyzed as in (A) above. D, WT or Isa1-depleted (Isa1↓) mitochondria (200 μg of proteins) were supplemented with [³⁵S]cysteine, nucleotides, iron and as indicated, purified Isa1 precursor protein (pIsa1; 1 μg). Samples were incubated at 30 °C for 30 min and subsequently analyzed as in (A) above. Grx5, glutaredoxin 5; pGrx5, Grx5 precursor protein.

Figure 3

Effects of Grx5 depletion or Isa1 depletion on [2Fe-2S] cluster loading of newly imported ferredoxin (Yah1) in isolated mitochondria.A, WT or Grx5↓ mitochondria (200 μg of proteins) were supplemented with [³⁵S]cysteine, nucleotides, iron and as indicated, ferredoxin precursor protein (pYah1; 1X = 0.5 μg) (40). Samples were incubated at 30 °C for 30 min, centrifuged, and mitochondrial pellets (“P”) were analyzed by native PAGE, followed by autoradiography. B, assays were performed with WT and Isa1↓ mitochondria under identical conditions as in (A) above. Grx5, glutaredoxin 5; pYah1, Yah1 precursor protein.

Figure 4

Presence of Grx5, but not ISA components, in mitochondria needed for promoting [2Fe-2S] cluster assembly in isolated WT cytoplasm.A, mitochondria (WT or Grx5↓; 200 μg of proteins) were mixed with isolated WT cytoplasm (200 μg of proteins), and reaction mixtures were supplemented with [³⁵S]cysteine (10 μCi), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), iron (10 μM) and as indicated, apo-ΔNYah1 protein (1 μg) (42). Samples were incubated at 30 °C for 30 min and centrifuged. The cytoplasm/supernatant (“S”) fractions thus obtained were analyzed by native PAGE, followed by autoradiography. B, mitochondria (WT or Isa1↓; 1X = 100 μg of proteins) were mixed with WT cytoplasm (200 μg of proteins), and reaction mixtures were supplemented with [³⁵S]cysteine, nucleotides, iron, and apo-ΔNYah1 protein. Assays were performed as in (A) above. C, mitochondria (WT or Isa2↓; 200 μg of proteins) were mixed with WT cytoplasm (200 μg of proteins), and reaction mixtures were supplemented with [³⁵S]cysteine, nucleotides, iron and as indicated, apo-ΔNYah1 protein. Assays were performed as in (A) above. D, mitochondria (WT or Iba57↓; 200 μg of proteins) were mixed with WT cytoplasm (200 μg of proteins). Samples were supplemented with [³⁵S]cysteine, nucleotides, iron and as indicated, apo-ΔNYah1 protein. Assays were performed as in (A) above. ΔNYah1, N-terminal 60 amino acids including the mitochondrial targeting signal removed from the Yah1 precursor protein (pYah1); Grx5, glutaredoxin 5.

Figure 5

Mitochondria lacking Grx5 cannot promote Leu1 [4Fe-4S] cluster assembly in cytoplasm.A, cytoplasm was isolated from WT, Gal-Grx5 repressed (Grx5↓), Gal-Isa1 repressed (Isa1↓), and LEU1 gene deleted (Δleu1) strains, and endogenous Leu1 isopropylmalate isomerase activity was measured in samples containing 200 μg of proteins (43, 44). Data shown are the means ± SD (n = 4). B, isolated mitochondria (WT, nfs1, Ssq1↓, Grx5↓, Isa1↓ or Isa2↓; 200 μg of proteins) were mixed with isolated Δleu1 cytoplasm (200 μg of proteins) and apo-Leu1^R protein (2 μg). The Δleu1 cytoplasm alone or apo-Leu1^R protein alone served as background controls. All reaction mixtures were supplemented with unlabeled cysteine (10 μM), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), and iron (10 μM) and incubated at 30 °C for 30 min. After removal of mitochondria by centrifugation, the resulting cytoplasm/supernatant fractions were assayed for reconstituted Leu1^R isopropylmalate isomerase activity as in (A) above. Mutant nfs1, hypomorphic allele of Nfs1; Ssq1↓, Gal-Ssq1 repressed (42, 57). Data shown are the means ± SD (n = 4). Leu1^R, recombinant Leu1; Grx5, glutaredoxin 5.

Figure 6

Mitochondria lacking Grx5 cannot activate defective cytoplasm in the nfs1 mutant required for reconstitution of cytoplasmic Fe-S cluster assembly.A, mitochondria and cytoplasm were isolated from WT and nfs1 strains and as indicated, they were mixed in different combinations. Samples were supplemented with [³⁵S]cysteine (10 μCi), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), iron (10 μM), and apo-ΔNYah1 protein (1 μg) and incubated at 30 °C for 30 min. Mitochondria were removed from assay mixtures by centrifugation, and the resulting cytoplasm/supernatant (“S”) fractions were analyzed by native PAGE, followed by autoradiography. B, mitochondria (WT, Grx5↓, Isa1↓, or Isa2↓; 200 μg of proteins) were mixed with nfs1 cytoplasm (200 μg of proteins) and apo-ΔNYah1 protein (1 μg). Samples were supplemented with [³⁵S]cysteine, nucleotides, and iron, and then incubated at 30 °C for 30 min. After centrifugation, the cytoplasm/supernatant (“S”) fractions were analyzed as in (A) above. The vertical dividing line (between lanes 2 and 3) indicates removal of an unnecessary sample from the same autoradiograph. C, as indicated, mitochondria (nfs1, WT, Grx5↓, or Isa1↓; 200 μg of proteins) were mixed with cytoplasm isolated from the nfs1 strain (200 μg of proteins) and apo-Leu1^R protein (2 μg). Reaction mixtures were supplemented with unlabeled cysteine (10 μM), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), and iron (10 μM) and incubated at 30 °C for 30 min. Mitochondria were removed by centrifugation, and the resulting cytoplasm/supernatant fractions were assayed for reconstituted Leu1^R isopropylmalate isomerase activity as in Figure 5B. Data shown are the means ± SD for four biological replicates. ΔNYah1, N-terminal 60 amino acids including the mitochondrial targeting signal removed from the Yah1 precursor protein (pYah1); Leu1^R, recombinant Leu1; Grx5, glutaredoxin 5; Fe-S, iron-sulfur.

Figure 7

Correcting inability of Grx5↓ mitochondria to promote cytoplasmic Fe-S cluster assembly by importing purified Grx5 protein.A, mitochondria lacking endogenous Grx5 (Grx5↓; 200 μg of proteins) were mixed with WT cytoplasm (200 μg of proteins), and assay mixtures were supplemented with [³⁵S]cysteine, nucleotides, and iron. As indicated, the Grx5 precursor protein (pGrx5; 1 μg) and/or ΔNYah1 protein (1 μg) were added and samples were incubated at 30 °C for 30 min. Mitochondria were removed by centrifugation and the resulting cytoplasm/supernatant (“S”) fractions were analyzed by native PAGE, followed by autoradiography. B, mitochondria (WT or Grx5↓; 200 μg of proteins) were mixed with Δleu1 cytoplasm (200 μg of proteins) and apo-Leu1^R protein (2 μg). The pGrx5 protein (1 μg) was included as indicated. Apo-Leu1^R alone served as the background control. All reaction mixtures were supplemented with unlabeled cysteine (10 μM), nucleotides (4 mM ATP, 1 mM GTP, 2 mM NADH), and iron (10 μM) and incubated at 30 °C for 30 min. After removal of mitochondria by centrifugation, and the cytoplasm/supernatant fractions were assayed for reconstituted Leu1^R isopropylmalate isomerase activity as in Figure 5B. Data shown are the means ± SD (n = 4). ΔNYah1, N-terminal 60 amino acids including the mitochondrial targeting signal removed from the Yah1 precursor protein (pYah1); Leu1^R, recombinant Leu1; Grx5, glutaredoxin 5; Fe-S, iron-sulfur.

Figure 8

Trafficking role of Grx5 in cellular Fe-S protein biogenesis.A, summary of the results presented here. For all biosynthetic processes tested, the efficiency of WT mitochondria was arbitrarily considered 100% as indicated by four pluses (“++++”). ND, not determined. B, a model for Grx5 acting at the focal point of “three-way” Fe-S cluster trafficking in mitochondria. In the conventional mitochondrial pathway, the ISC machinery synthesizes [2Fe-2S] and [4Fe-4S] clusters for organellar iron proteins (16, 20). The core components of the machinery are also involved in producing two different intermediates: S_int for cytoplasmic tRNA thiolation and (Fe-S)_int for cytoplasmic Fe-S cluster assembly (42). The ISC pathway begins with the activity of a protein complex consisting of Nfs1 and other factors (not shown). The Nfs1 cysteine desulfurase abstracts sulfur from the amino acid cysteine to form a persulfide sulfur intermediate, which is then donated to the Isu1/2 scaffold. At this stage, this sulfur may leave the conventional ISC pathway and is utilized for S_int formation in a process that does not require downstream components such as the Ssq1 chaperone (42), Grx5 (Fig. 4A), or the ISA complex (Fig. 4, B–D). As needed, the S_int is exported to the cytoplasm where it is utilized for thiolation of tRNAs (42, 53, 54). In the context of Fe-S cluster assembly, however, the conventional ISC pathway continues with Isu1/2 forming a [2Fe-2S] cluster intermediate by combining the persulfide sulfur from Nfs1 and iron from an undetermined source. Ssq1 (together with other proteins; not shown) then promotes transfer of this intermediate to Grx5 (16, 20). At this stage, Grx5 controls “three-way” trafficking of Fe-S clusters/intermediates. First, the [2Fe-2S] cluster may be directly transferred from Grx5 to a recipient such as ferredoxin (Yah1), with no further requirement of any other downstream ISC components such as the ISA complex (Fig. 3). Second, Grx5 may transfer [2Fe-2S] cluster to the ISA complex, which generates a [4Fe-4S] cluster by reductive coupling for aconitase. The aconitase [4Fe-4S] cluster assembly is blocked in mitochondria lacking Grx5 or Isa1 (Fig. 1), but the biosynthetic process is efficiently restored by newly imported Grx5 or Isa1 into respective mutant mitochondria (Fig. 2, A–D). Finally, Grx5 also plays a vital role for (Fe-S)_int synthesis. Here, the pathway branches again. The (Fe-S)_int moves from Grx5 to the Atm1 transporter and is exported to the cytoplasm. Once exported, (Fe-S)_int is utilized by the CIA machinery, generating cytoplasmic Fe-S clusters (42). Grx5-depleted mitochondria cannot promote [2Fe-2S] or [4Fe-4S] cluster assembly (Figs. 4 and 5, respectively) but can efficiently do so with the help of newly imported Grx5 (Fig. 7). Notably, both iron and sulfur for cytoplasmic Fe-S cluster assembly likely originate from the mitochondria (42), revealing an essential and direct role of Grx5 in (Fe-S)_int synthesis and trafficking. CIA, cytoplasmic iron-sulfur protein assembly machinery; Fe-S, iron-sulfur; (Fe-S)_int, (Fe-S) intermediate; Grx5, glutaredoxin 5; IPM, isopropylmalate; ISC, mitochondrial iron-sulfur cluster.