Using the ability to cultivate frataxin/frh-1 null mutant C. elegans in hypoxia, we performed a forward genetic selection for rare suppressor mutations that allow frh-1 null mutant animals to grow at what is normally a non-permissive oxygen tension (Fig. 1a). frh-1(tm5913) animals carrying a deletion in the frataxin gene were grown at 1% oxygen, a permissive condition under which they are viable, and randomly mutagenized, generating hundreds of new mutations throughout the genome in each of thousands of animals. Two generations later, tens of thousands of F 2 progeny were transferred to 10% oxygen and challenged to grow at this non-permissive oxygen tension for a frh-1 null mutant. Whereas most animals arrested development as larvae, such as the parental frh-1(tm5913) strain, rare animals carrying a newly induced suppressor mutation reached adulthood and were isolated. These mutants still carried a deletion in the frataxin gene. Following whole-genome sequencing, we identified that suppressor mutant strains also carried missense mutations in one of two genes: the cysteine desulfurase nfs-1 (one allele corresponding to R244K) or the mitochondrial ferredoxin Y73F8A.27 (three independent alleles corresponding to E117K, A126V and P127S) (Fig. 1b and Extended Data Table 1).
Fig. 1: Mutations in NFS1/nfs-1 or FDX2/fdx-2 partially rescue the growth defect caused by frataxin loss in C. elegans. a, A forward genetic screen using random chemical mutagenesis revealed particular substitution mutations in fdx-2 and nfs-1 that can rescue the loss of frataxin. b, Multiple sequence alignment of NFS1 (C. elegans residues 239–251) and FDX2 (C. elegans residues 117–127) including homologues from mammals, fish and invertebrates made using ClustalW. c, Synchronized frataxin-null animals grown at 7% oxygen for 4 days with or without suppressor mutations in fdx-2 and nfs-1. Scale bar, 1 mm. d–f, Growth of C. elegans at 7% oxygen (4 days) (d), 1% or 10% oxygen (2 days) (e) or 7% oxygen (2 days) (f) quantified by body length measurements. The number of individual worms in each group was: all groups n = 12 (d), all groups n = 12–14 (e), frh-1 n = 27, frh-1; nfs-1 n = 22, frh-1; nfs-1/+ n = 8, frh-1; fdx-2 n = 17, frh-1; fdx-2/+ n = 8 (f). For all panels, statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s (d,f) or Sidak’s (e) multiple comparison test. Error bars represent mean ± s.d. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Full size image
Most metazoa encode two ferredoxin paralogues, FDX1 and FDX2, both of which harbour [2Fe–2S] clusters and are localized to mitochondria. FDX2 is required for de novo [2Fe–2S] cluster biosynthesis, donating electrons to a persulfide on the scaffold protein ISCU2 (refs. 4,17,18). FDX1 has long been known to be required for steroidogenesis and, more recently, has been shown to be key for the synthesis of haem a (for cytochrome c oxidase) and lipoyl synthase19,20,21,22. These ferredoxins cannot functionally complement each other due to sequence divergence that lends specificity to target substrates18,19,20,21. Although basal metazoa such as sponges and anemones encode both paralogues, C. elegans contain only one mitochondrial ferredoxin, Y73F8A.27, and through phylogenetic analysis we inferred that C. elegans retain FDX2 and have lost FDX1 (Extended Data Fig. 1a). Thus, Y73F8A.27 has been renamed fdx-2. The alignment of C. elegans FDX-2 with human FDX2 and FDX1 revealed that C. elegans FDX-2 contains key sequence features19 of both FDX2 and FDX1 (Extended Data Fig. 1b), raising the possibility that it has acquired new FDX1-like functionality. Notably, all identified nfs-1 and fdx-2 suppressor mutations occur in highly conserved residues also present in human NFS1 and FDX2, respectively (Fig. 1b and Extended Data Table 1).
We used CRISPR–Cas9 to generate nfs-1 and fdx-2 mutant alleles in a clean genetic background and confirmed that nfs-1(R244K), fdx-2(E117K), fdx-2(A126V) or fdx-2(P127S) mutations improved the growth of frh-1 null animals at non-permissive 7–10% oxygen (Fig. 1c–e and Extended Data Fig. 1c), which is the natural environmental oxygen tension for C. elegans nematodes23. Analysing frataxin-null animals carrying heterozygous mutations in nfs-1(R244K) or fdx-2(A126V) revealed that these suppressor mutations dominantly rescue growth of frataxin mutants (Fig. 1f). The point mutations in nfs-1 and fdx-2 also significantly improved the growth of frh-1 mutants at the more severe 21% oxygen (Extended Data Fig. 1d). In a frh-1(wt) background the nfs-1(R244K) and fdx-2(A126V) point mutations caused no growth defect at 21% oxygen (Extended Data Fig. 1e), whereas the fdx-2(E117K) and fdx-2(P127S) point mutations produced small but significant effects on growth rate. At 1% oxygen, a permissive condition that supports the growth and fertility of frataxin mutants7, the nfs-1 and fdx-2 suppressor mutations further rescued the frh-1 growth rate to wild-type levels (Fig. 1e and Extended Data Fig. 1f). Taken together, these results identify suppressor mutations within the Fe–S cluster assembly complex that partially rescue the growth and developmental defects of frataxin loss.
To understand how the nfs-1 and fdx-2 suppressor mutations rescue the growth defects of frataxin-null animals, we first analysed transcriptional reporters for C. elegans stress responses. Both the hsp-6::gfp reporter for mitochondrial stress and the gst-4::gfp reporter for oxidative stress were elevated in the frh-1 mutant at 21% oxygen but not induced at 1% oxygen7 (and Extended Data Fig. 2a,b). The striking rescue by hypoxia may result from increased Fe–S cluster production and stability7,24, as well as the ability of hypoxia to rescue downstream pathways dependent on Fe–S clusters such as the ETC25,26,27 and lipoylation20. Similarly, induction of hsp-6::gfp fluorescence, which is a sensitive readout of mitochondrial membrane potential and protein import efficiency28, was partially decreased by fdx-2 and nfs-1 mutation in the frh-1 null background (Fig. 2a and Extended Data Fig. 2a), suggesting an improvement in mitochondrial integrity. By contrast, the fdx-2 and nfs-1 suppressor mutations did not change the induction of gst-4::gfp fluorescence (Extended Data Fig. 2b), arguing against a global hypoxia-like restoration of ISCs to wild-type levels.
Fig. 2: Frataxin suppressor mutations restore levels of Fe–S cluster-containing ETC complexes. a, Mean intestinal fluorescence of age-matched day 1 adult animals containing hsp-6::gfp exposed to 21% oxygen for 24 h. The number of individual worms in each group was n = 11–12. b, Quantitative TMT proteomics of complex I subunits in wild-type animals, frataxin mutants and frataxin mutants with suppressor mutations nfs-1(R244K) or fdx-2(A126V) grown continuously at 1% oxygen. Values are normalized to wild type; each line represents one protein from n = 1 experiment. Proteins shown from which at least two peptides were quantified. c, Growth of C. elegans at 1% or 10% oxygen for indicated durations. The number of individual worms in each group was n = 20. For all panels, statistical significance was calculated using one-way ANOVA followed by Sidak’s multiple comparison test (a,c). Error bars represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. a.u., arbitrary units. Full size image
To identify compromised cellular processes in the frataxin mutant that are rescued by nfs-1 or fdx-2 mutation, we performed quantitative tandem mass tag (TMT) proteomics in animals incubated continuously at 1% oxygen or animals shifted from 1% to 21% oxygen for 2 days. Analysis of Fe–S cluster-containing proteins revealed that C. elegans frataxin mutants shifted from 1% to 21% oxygen tended to be depleted for [4Fe–4S] proteins whereas [2Fe–2S] proteins were largely unaffected (Extended Data Fig. 3a and Supplementary Table 1). Following prolonged (10-day) loss of frataxin in cell culture virtually all Fe–S cluster proteins are depleted29. The C. elegans proteomics may reflect differences in half-life between [4Fe–4S]-containing and [2Fe–2S]-containing proteins after 2 days of Fe–S cluster biogenesis interruption. Of the 23 Fe–S cluster-containing proteins whose levels were depleted by at least 1.5-fold, hypoxia partially increased the levels of 21 out of 23 proteins, whereas the fdx-2(A126V) and nfs-1(R244K) suppressor mutations restored levels of only 10 Fe–S containing proteins, which included subunits of ETC complex I and complex II (Extended Data Fig. 3a).
In C. elegans, mutations in core subunits of complex I can destabilize the entire complex, resulting in the loss of other individual proteins27. Indeed, we found that all complex I subunits (except for the acyl carrier protein NDUFAB1) were depleted in frh-1 mutants and rescued by fdx-2 or nfs-1 suppressor mutations (Fig. 2b). We confirmed these proteomics results with western blots against complex I subunit NDUFS3 (Extended Data Fig. 3b,c), which showed that at 1% or 21% oxygen frataxin loss caused low levels of complex I that were partially rescued by fdx-2 or nfs-1 point mutation. To determine whether ETC rescue was sufficient to improve the growth of frataxin mutants we expressed the yeast NADH dehydrogenase NDI1 (ref. 30), a single polypeptide that can bypass complex I and complex II in C. elegans27,31,32. By conveying electrons from NADH directly to ubiquinone, NDI1 is able to restore NADH redox balance as well as oxygen consumption and proton pumping by complexes III and IV. Expression of NDI1 was sufficient to partially rescue the growth of frh-1 mutants (Fig. 2c). Taken together, these results indicate fdx-2 and nfs-1 point mutations partially restore Fe–S cluster biosynthesis in the absence of frataxin, and that the resulting increased flux through the ETC may be paramount to the rescue of animal growth and development.
To determine whether the nfs-1 and fdx-2 suppressor mutations were acting through a common mechanism we tested their genetic interaction. A double mutant of the C. elegans mutations nfs-1(R244K); fdx-2(A126V) in a frataxin wild-type background was synthetic sterile and grew slowly (Fig. 3a), despite each suppressor mutation on their own having no growth defect (Extended Data Fig. 1e). The nfs-1(R244K); fdx-2(A126V) double mutant also showed reduced lipoic acid staining (Extended Data Fig. 4a), consistent with reduced Fe–S cluster biosynthesis. The slow growth, sterility and low lipoic acid staining of the nfs-1(R244K); fdx-2(A126V) double mutant was rescued by hypoxia (Fig. 3a and Extended Data Fig. 4a), similar to other Fe–S cluster-deficient mutants and further supporting a loss of Fe–S cluster biosynthesis. Together, these results are consistent with the nfs-1 and fdx-2 suppressor mutations acting through the same pathway to rescue frataxin loss.
Fig. 3: Excess FDX2 is detrimental to NFS1 activity and Fe–S cluster biosynthesis. a, Growth of animals for 3 days at room temperature exposed to 21% or 1% oxygen. The number of individual worms in each group was n = 20. b, Cryo-EM structure of the human Fe–S cluster assembly complex containing FDX2 (ref. 9) (Protein Data Bank 8RMC) with boxes indicating homologous residues to C. elegans suppressor mutations. c, Cysteine desulfurase activities of SDA ec (0.5 µM; NFS1–ISD11–ACP ec ) complexes containing ISCU2 (1.5 µM; white) and increasing equivalents of FXN (0.25–30 µM; grey), FDX2 ox (0.25–30 µM, oxidized ferredoxin 2; blue) and FDX2 ox E131K (0.25–30 µM; purple). Reactions were initiated with 2 mM l-cysteine, quenched after 3 min and the sulfide was converted to methylene blue to determine activities. For all groups n = 3 independent experiments. d, Iron–sulfur assembly activities on ISCU2 (100 µM) for complexes containing SDA ec (1 µM), FXN (0–60 μM), FDX2 or FDX2 E131K (0–60 μM), FDXR (1 μM) and NADPH (500 μM). Reactions were initiated with 500 μM l-cysteine, and the change in ellipticity at 430 nm was fit to a linear equation to determine the rate of Fe–S assembly on ISCU2. The numbers on the x axis represent protein equivalents compared with SDA ec . ND, not detectable. For all groups n = 3 independent experiments. The initial and final circular dichroism spectra and averaged change in ellipticity at 430 nm for determining the reaction rate for each experiment are shown in Extended Data Fig. 5a–c. e, In wild-type conditions frataxin and FDX2 compete for binding to NFS1, promoting persulfide transfer and donating electrons, respectively, at distinct steps in the biosynthesis of Fe–S clusters. In the context of low or zero frataxin, or FDX2 overexpression, Fe–S synthesis is blocked due to frataxin displacement by FDX2 and/or FDX2 directly inhibiting NFS1 activity. Point mutations that weaken the NFS1–FDX2 interaction, or simply lowering FDX2 levels to generate NFS1 unbound by FDX2, can partially restore Fe–S synthesis and support growth in C. elegans. f, Three-day growth assay with K562 or 293T cells overexpressing (O/E) GFP or FDX2 cDNA, grown in 21% or 1% oxygen tensions. Experiments were conducted in three biological replicates in technical duplicate for a total n = 6. g, Immunoblots for FDX2, lipoic acid, OXPHOS, NFS1 and loading control tubulin in K562 (left) and 293T (right) cell lysates collected from f. For gel source data, see Supplementary Data 1. For western blotting n = 3 biological replicates; all individual replicates for lipoic acid are shown and quantified in Supplementary Data 2. Statistical significance was calculated using one-way (a) or two-way (f) ANOVA followed by Sidak’s multiple comparison test. Error bars represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Full size image
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