Human Molecular Genetics, 2002, Vol. 11, No. 21 2635-2643
© 2002 Oxford University Press
A non-essential function for yeast frataxin in iron–sulfur cluster assembly
1Unité de Biochimie Physiologique, Université Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium and 2lnstitut für Physiologische Chemie der Universität München, Butenandtstrasse 5, 81377 München, 80336, Germany
Received June 14, 2002; Accepted July 30, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Friedreich's ataxia is caused by a deficit in frataxin, a small mitochondrial protein of unknown function that has been conserved during evolution. Previous studies have pointed out a role for frataxin in mitochondrial iron–sulfur (Fe–S) metabolism. Here, we have analyzed the incorporation of Fe–S clusters into yeast ferredoxin imported into isolated energized mitochondria from cells grown in the presence of glycerol, an obligatory respiratory carbon source. Similar amounts of apo-ferredoxin precursor were imported into mitochondria and processed in wild-type and yfh1-deleted (
YF111) strains. However, the incorporation of Fe–S clusters into apo-ferredoxin was significantly reduced in YFH1 mitochondria. The newly assembled ferredoxin was stable, excluding the possibility that the decreased incorporation was a result of increased oxidative damage. When YFH1 cells were grown in raffinose medium, the formation of holo-ferredoxin was low, as a consequence of the decrease in ferredoxin precursor import into mitochondria. However, the decrease in the conversion rate of apo- into holo-ferredoxin was in the same range as for glycerol-grown cells, indicating that the extent of the defect in Fe–S protein assembly is similar under different physiological conditions. These data show that frataxin is not essential for Fe–S protein assembly, but improves the efficiency of the process. The large variations observed in the activity of Fe–S cluster proteins under different physiological conditions result from secondary defects in the physiology of YFH1 cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Iron–sulfur (Fe–S)-containing proteins are ubiquitous in living organisms. The synthesis of Fe–S clusters is a complex process involving more than 10 proteins that have been conserved from bacteria to humans (1,2). In Saccharomyces cerevisiae, Fe–S clusters are made within mitochondria, and are probably exported into the cytosol by the ABC transporter Atm1p (3). The mechanisms by which Fe–S clusters are synthesized and incorporated into proteins in vivo are not completely understood. A pyridoxal phosphate-dependent cysteine desulfurase (Nfs1p in S. cerevisiae) provides elemental sulfur (4) to a machinery composed of several proteins acting in concert to assemble the Fe–S clusters. In yeast, Isu1p, Isu2p, the chaperones Ssq1p and Jac1p, the Yah1p ferredoxin, and Isa1p and Isa2p participate in Fe–S cluster biosynthesis (for a review, see 2). An important issue to address is whether frataxin participates in this process.
Frataxin is a small hydrophilic protein of unknown function that has been conserved during evolution from Gram-negative bacteria to humans (5), and in eukaryotes it localizes to mitochondria (6–8). Frataxin does not bear homology to other proteins and is characterized by a novel fold consisting of a five-stranded antiparallel ß-sheet platform supported by two 
-helices (9–11). In humans, a reduction in frataxin concentration causes Freidreich's ataxia, a recessive neurodegenerative disease associated with cardiomyopathy (12). Specific defects in Fe–S-containing enzymes (13) and mitochondrial iron deposits have been observed in endomyocardial biopsies from patients (14). Mouse models of Friedreich's ataxia have led to the conclusion that the decline in Fe–S protein activity occurs prior to mitochondrial iron accumulation, suggesting that iron toxicity is not the primary cause of the disease (15). Loss of frataxin in transgenic mice leads to early embryonic lethality (16), strongly suggesting that frataxin is also essential in humans.
In yeast, the deletion of YFH1, the yeast frataxin homologue gene, elicits a 10–15-fold increase in mitochondrial iron concentration in glucose-grown cells (17,18), and the accumulated iron cannot be extruded from mitochondria (19). This results in a profound alteration in cellular iron homeostasis, as shown by the increased expression of all the genes involved in the mobilization of iron from the external medium and intracellular stores (6,20). Yfh1p deficiency also results in low activity of Fe–S proteins such as aconitase and succinate dehydrogenase (13,17), and increased sensitivity to hydrogen peroxide and high iron concentrations (6,18). In contrast, no mitochondrial iron overload is observed in cells grown in glycerol, a non-fermentable carbon source; yet they still exhibit decreased aconitase activity (21). Altogether, these data show that it is essential to distinguish primary effects from secondary causes in the phenotypes observed in frataxin-deficient strains.
It has been shown that yeast frataxin expressed in Escherchia coli is a monomer that does not bind iron; however, in vitro and in the presence of a large iron excess, yeast frataxin becomes a multimer binding 3000 atoms of iron (22). Recombinant human frataxin binds 
10 atoms of iron per subunit (23). However, iron binding by frataxin in vivo under physiological conditions has still to be demonstrated. On the basis that frataxin has the same evolutionary lineage as HscA and HscB, two chaperones participating in Fe–S cluster synthesis, a role for frataxin in Fe–S cluster metabolism has been predicted (24). Moreover, it has recently been reported that the incorporation of Fe–S cluster into Yah1p ferredoxin was severely impaired in a YFH1 strain (25). In the assay used by the authors, apo-ferredoxin was imported into isolated mitochondria, and the conversion of the newly imported apo-ferredoxin to the holo-form was measured. However, the YFH1 strain used in this work (FKEN015-01A, http://mips.gsf.de/proj/yeast/CYGD/db/index.html) was a rho- mutant and therefore had to be cultivated in the presence of a fermentable carbon source, which leads to mitochondrial iron overload. To avoid secondary effects associated with frataxin deficiency, we decided to reinvestigate this study using another strain with a stable mitochondrial genome and grown in the presence of glycerol. Our data led to the conclusion that yeast frataxin is not essential for the incorporation of Fe–S clusters into ferredoxin. However, it improves the efficiency of the reaction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Protein import into mitochondria of
YFH1 cellsThe efficiency of protein import into mitochondria was compared for strains grown in glycerol, an obligatory carbon source, and raffinose, a non-repressible fermentable sugar. As previously reported, no increase in the production of rho- mutants was observed in the presence of these carbon sources, and thus the mitochondrial genome of our
YFH1 strain was perfectly stable (18,21, and data not shown). The precursor of subunit 4 of cytochrome c oxidase, Cox4p, was radiolabeled in vitro with [35S]methionine and used to monitor protein import into isolated energized mitochondria. The same levels of mature Cox4p were detected (Fig. 1A) in the mitochondria of wild-type and YFH1 cells grown in glycerol medium, indicating that import was equally efficient in wild-type and mutant strains. Mitochondrial protein import was also quite efficient in the wild-type strain grown in raffinose medium; however, a severe decrease in import was observed in the YFH1 strain (Fig. 1A), which is most likely explained by the low membrane potential measured in isolated mitochondria with the DiSC3(5) dye (data not shown).
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Mitochondrial iron concentration was similar in wild-type and
YFH1 cells grown in glycerol medium, and the ratio of aconitase to isocitrate dehydrogenase activity was reduced by 35% (Fig. 1B and D). The aconitase concentration was the same in wild-type and YFH1 mitochondria (Fig. 1C), implying that the decrease in aconitase activity resulted from a loss of function. When cells were grown in raffinose medium supplemented with 2 µM FeSO4, compared with wild-type, YFH1 mitochondria exhibited a 5-fold increase in iron concentration and low aconitase activity (Fig. 1B and D). Although substantial, the decrease in aconitase concentration (Fig. 1C) could not alone explain the loss of aconitase activity. The latter most likely resulted from the absence, or instability, of the active holo-aconitase form.
Incorporation of Fe–S clusters into radiolabeled ferredoxin imported into mitochondria of glycerol-grown cells
35S-radiolabeled apo-ferredoxin precursor was imported into energized mitochondria for 4 minutes and the import reaction was stopped by addition of valinomycin, a potassium ionophore that dissipates the membrane potential. Mitochondria were further incubated to enable the incorporation of Fe–S clusters into newly imported ferredoxin, and aliquots were taken up after 4, 8, 16 and 32 minutes. The conversion of mature apo-ferredoxin into the holo form was analyzed by native-gel electrophoresis and autoradiography (Fig. 2A). The majority of the mitochondrial proteins, including the ferredoxin precursor, penetrate this gel poorly. However, the mature form of ferredoxin, which is very acidic, gives three bands of different mobility corresponding to a fast-migrating holo-ferredoxin species and two slowly migrating reduced and oxidized apo forms (26). The oxidized apo-ferredoxin species cannot incorporate an Fe–S cluster. As observed for Cox4p, the import of ferredoxin into wild-type and YFH1 mitochondria was similar (Fig. 2B). During the import reaction, apo-ferredoxin was rapidly converted into holo-ferredoxin in wild-type and YFH1 mitochondria (Fig. 2C). In the second phase, conversion of apo- to holo-ferredoxin was slowed down, and, compared with wild-type, holo-ferredoxin formation was reduced by 20% in YFH1 mitochondria. We verified that the reaction was inhibited by o-phenanthroline, an iron chelator (Fig. 2D). Therefore, frataxin deficiency only slightly decreased the efficiency of Fe–S cluster assembly. For comparison, we decided to perform the same experiment with a ISU1 strain. Isu1p, in concert with its isoform Isu2p, plays an essential role in Fe–S cluster assembly (27,28), and a ISU1ISU2 strain is not viable. However, in a ISU1 strain, Isu2p can partially substitute for Isu1p, and thus a ISU1 strain is viable (28). We found YFH1 and ISU1 phenotypes to be quite similar regarding mitochondrial iron overload and decline in aconitase activity in glycerol grown cells (Figs 1A–C and 3A–C). There was no significant decrease in the rate of Fe–S incorporation into 35S-radiolabeled ferredoxin (Fig. 3D and F). These data show that frataxin deficiency elicits a partial defect in Fe–S cluster assembly that is stronger than in a ISU1 strain.
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Incorporation of 35S from cysteine into imported ferredoxin expressed in E. coli
In the experiments described above, minute amounts of radiolabeled ferredoxin were used, raising the possibility that the apo protein was rapidly converted into the holo form by saturating amounts of the Fe–S cluster assembly machinery, even though the activity of the latter was not optimal. In a second experimental approach, chemical amounts (5 µg) of the purified apo-ferredoxin precursor tailed with a six-histidine tag and expressed in E. coli were used, and the elemental sulfur used for Fe–S cluster assembly was provided by [35S]cysteine added in the import mixture. Under these conditions, excess apo-ferredoxin was used. It was first verified that the amount of [35S]cysteine imported into wild-type and
YFH1 mitochondria was similar (data not shown). The import of the ferredoxin precursor was estimated after 30 and 60 minutes of incubation by western blotting using a six-histidine antibody. The amount of mature ferredoxin recovered in mitochondria was identical for wild-type, YFH1 and ISU1 strains (Fig. 4A and B). The holo-ferredoxin form was estimated in native gels by the amount of 35S incorporated into the Fe–S cluster of the newly imported ferredoxin. While the amount of 35S incorporated into holo-ferredoxin in wild-type mitochondria increased with incubation time, even after 60 minutes of incubation, [35S]holo-ferredoxin was barely or not detectable in YFH1 mitochondria and was dramatically decreased in ISU1 mitochondria (Fig. 4C and D). These data show that, in the presence of saturating amounts of the ferredoxin substrate, the activity of the Fe–S cluster assembly machinery in ISU1 and YFH1 mitochondria is a limiting factor for the conversion of chemical amounts of apo-ferredoxin into the holo form. Moreover, we confirm here that the defect is more pronounced in the YFH1 than in the ISU1 strain.
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Holo-ferredoxin is stable in a
YFH1 strainDecrease in holo-ferredoxin formation can result either from impaired Fe–S cluster incorporation into ferredoxin or from increased instability of the newly incorporated cluster. Import of the [35S]apo-ferredoxin precursor was carried out in wild-type and
YFH1 mitochondria for 4 minutes and stopped by valinomycin. The mitochondria were washed twice and resuspended in a new buffer without respiratory substrates, and aliquots were taken up after 30, 60 and 90 minutes. The amount of [35S]holo-ferredoxin remained constant after transfer into new buffer (Fig. 5A and C). Therefore, in glycerol-grown strains, the newly synthesized holo-ferredoxin was stable, excluding increased oxidative damage in YFH1 mitochondria. This conclusion was also supported by the observation that aconitase activity was extremely stable in YFH1 mitochondria even after an 80-minute incubation at 30°C (data not shown).
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Incorporation of Fe–S cluster in apo-ferredoxin of
YFH1 cells grown in raffinoseThe conversion of [35S]apo-ferredoxin into the holo form in mitochondria of raffinose-grown cells was measured as reported above. As for Cox4p, the import of the ferredoxin precursor into mitochondria was dramatically decreased in the
YFH1 strain (Fig. 6B). Although the very low amount of imported precursor decreased the accuracy in the measurement of the conversion of apo- to holo-ferredoxin, a signal corresponding to the holo form of ferredoxin was detected in YFH1 mitochondria (Fig. 6A). The intensity of this signal increased with time, and, compared with wild-type, the ratio of holo- to total ferredoxin concentration was reduced by 20% after a 20-minute incubation (Fig. 6C). These data show that synthesis of Fe–S clusters does occur in isolated mitochondria of YFH1 cells grown in raffinose medium. The very low amount of holo-ferredoxin results from a drastically decreased protein import into isolated mitochondria.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous studies have suggested that frataxin deficiency alters iron homeostasis, defence against oxidative damage and activity of Fe–S proteins (6,13,17–20). What is the primary target? A striking feature of W303-1B yfh1 mutants is their contrastingly different phenotype depending on whether they are cultivated in fermentable or respiratory carbon sources (20,21). In the presence of fermentable substrates, mitochondrial iron overload is associated with increased sensitivity to oxidative stress and general alteration in iron homeostasis. The very low activity of Fe–S proteins may reflect the oxidation and release of their Fe–S clusters. However, in glycerol-grown cells, there is no increase in mitochondrial iron concentration, and respiration is high. Therefore, glycerol is a particularly suitable carbon source to assess the role played by frataxin in Fe–S protein assembly and to avoid side-effects such as iron-induced damage. Using two complementary approaches, we have shown here that in a frataxin-deficient strain, Fe–S clusters can be incorporated into ferredoxin imported into isolated mitochondria. Compared with wild-type, the incorporation of Fe–S clusters is reduced by
20% on average when minute amounts of radiolabeled apo-ferredoxin are imported, and much more severely when chemical saturating amounts of recombinant apo-ferredoxin are used. The newly assembled holo-ferredoxin is stable, excluding the possibility that under these conditions the decrease in holo-protein concentration results from increased release of damaged Fe–S clusters. In other words, the decrease results from a defect in Fe–S protein assembly and not from oxidative damage. This decrease is more severe in a YFH1 than in a ISU1 strain. In the latter, although the function of Isup proteins is essential in the assembly process, Isu2p can partially substitute for Isu1p.
We can conclude that yeast frataxin plays a role in the conversion of apo-ferredoxin into holo-ferredoxin, and thus, more generally, in Fe–S protein assembly. This role is not essential. However, a reduced conversion rate of apo- to holo-ferredoxin is the only defect observed in the mitochondria of frataxin-deficient cells grown in glycerol medium. These data strongly suggest that a defect in the assembly of Fe–S proteins is the primary cause of the dysfunctions of frataxin-deficient cells. Reduced Fe–S cluster incorporation can result from impaired catalytic activity of the enzymes involved in the process, or decreased availability of the iron or cysteine substrates. Since similar amounts of [35S]cysteine are imported into wild-type and YFH1 mitochondria, a deficit in cysteine is unlikely. On the other hand, it is not excluded that iron availability is limiting in YFH1 mitochondria. A recent paper (29) studying the assembly of Fe–S proteins in mitochondrial lysates under anerobic conditions reached the same conclusion; however, the effect of frataxin deficiency was more drastic. This is not contradictory to our own data. First, the assay used by the authors may detect the Fe–S protein assembly defects in a more sensitive manner than ours; for instance, the NFU1 deletion decreased Fe–S protein assembly by 40%, although no defect was detected in vivo (29). However, we think that the main reason for this apparent discrepancy is that glucose was used as a carbon source. Under these conditions, we have never been able to measure any aconitase activity in YFH1 cells, even in low-iron media, indicating that Fe–S protein assembly is greatly impaired. The origin of this severe defect may be multiple, including lack of unknown important cofactors of the reaction or poor bioavailability of the mitochondrial iron. Moreover, several proteins involved in Fe–S protein assembly are Fe–S proteins, and, therefore, might be not fully functional.
Frataxin might be a component of the Fe–S protein assembly machinery. Structural data predict a protein partner for frataxin interacting at the level of the ß-sheet platform. Therefore, frataxin might be a chaperone regulating the sequential assembly of iron and sulfur. On the other hand, yeast and bacterial frataxins bind iron in vitro (22,23,30). It has been shown that iron binding in E. coli frataxin is mediated by acidic residues located on a negatively charged surface ridge (30). Thus, frataxin might also be an iron-binding protein in vivo, regulating iron fluxes through the mitochondria. A derangement in mitochondrial iron homeostasis may be explained by a defect in iron binding as well as in Fe–S protein assembly. In fact, any event preventing iron efflux from mitochondria disturbs iron homeostasis (20,21).
Frataxin is dispensable in a number of organisms such as archaea and Gram-positive bacteria (5), whereas it is strictly present in the mitochondria of all eukaryotes, suggesting that during evolution its function has become vital for the cell. Curiously, in contrast with W303 and D273 strains, several other strains do not maintain their mitochondrial genome in the absence of frataxin (6,8,18), and therefore need a fermentable carbon source for growth. In these strains, it has generally been admitted that the defect in Fe–S protein assembly and loss of mitochondrial DNA are the result of oxidative damage (6). Similarly, in humans, a frataxin defect gives the same presentation as deficiency in vitamin E, an antioxidant molecule; and in mammalian cells, early antioxidant defence is impaired (31). Although mitochondrial iron accumulation has been shown to be a late event in frataxin-deficient transgenic mice (15), it cannot be excluded that the presence of minute amounts of ‘free iron’ in a non-physiological form in mitochondria is the cause of the oxidative damage. Obviously, more work is necessary to determine the exact role played by oxidative stress in Friedreich's ataxia pathogenesis. Yeast strains, such as W303 or D273, in which the degree of oxidative stress can easily be modulated, genetically and physiologically, may prove particularly useful to understand the relationship between Fe–S protein assembly and levels of reactive oxygen species in frataxin deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Strains and media
The Saccharomyces cerevisiae strains were W303-1B (Mat
ade2-1 leu2-3, 112 his3 ura3-1 trp1-1) and its isogenic derivatives, W303-1BYFH1 (Mat ade2-1 leu2-3, 112 his3 ura3-1 trp1-1 yfh1::kanR) and W303-1BISU1 (Mat ade2-1 leu2-3, 112 his3 ura3-1 trp1-1 isu1::URA3) (18; F. Foury and A. Ramazzotti, unpublished data). Cells were grown in raffinose synthetic medium (2% raffinose, 0.7% yeast nitrogen base Difco, 0.5% ammonium sulfate, a mixture of amino acids and the required auxotrophic supplements) in the presence of 2 µM FeSO4, or in glycerol medium (2% yeast extract KAT and 3% glycerol).
Mitochondrial import and synthesis of holo-ferredoxin
Mitochondria were prepared after spheroplast lysis as described previously (32). 35S-radiolabeled precursors were synthesized using an in vitro transcription/translation kit (TNT coupled reticulocyte lysate systems, Promega) and [35S]methionine. The primers 5'-CCGCTCGAGTAATACGACTCACTATAGGGCGACTAACAATACTCACCCATTTCG-3' and 5'-CGCGGATCCGTAAAAGAGAAACAGAAGGGC-3' were used to amplify COX4 under the control of the T7 RNA polymerase promoter, and YAH1 was expressed from the SP6 RNA polymerase promoter in the pGEM3–YAH1 plasmid (25). Mitochondrial import and incorporation of Fe–S clusters into 35S-labeled ferredoxin or purified recombinant ferredoxin expressed in E. coli were performed as reported previously (25).
Quantification of signals
The amount of mature 35S-labeled ferredoxin or Cox4p was determined by SDS–PAGE and autoradiography of the dried gels (Biomax MR film, KODAK, Rochester, NY). 35S- radiolabeled apo- and holo-ferredoxin or recombinant holo-ferredoxin with 35S-labeled Fe–S cluster were detected after acrylamide gel electrophoresis under native conditions and autoradiography. The amount of mature recombinant ferredoxin was determined after SDS–PAGE and western blotting using an anti-histidine antibody (Penta-His antibody, QIAGEN, Leusden, NL). The autoradiographies were scanned and the signals were quantified with the Molecular Analyst software 1.4.1 (Bio-Rad, Hercules, CA).
Miscellaneous methods
Aconitase and isocitrate dehydrogenase activities were measured by standard procedures. Non-heme and non-Fe–S mitochondrial iron was measured using bathophenanthroline disulfonic acid (BPS) as described previously. The percentage of rho- mutants present in a culture was measured as previously reported (18,21) by spreading cells for single colonies on glucose-rich medium and counting the small white colonies that cannot grow on glycerol, or after 2,3,5-triphenyltetrazolium chloride overlay.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Belgian National Fund for Scientific Research, the European Commission, and the Interuniversity Poles of Attraction Program of the Belgian Government Office for Scientific, Technical and Cultural Affairs.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +32 10474691; Fax: +32 10473872; Email: foury{at}fysa.ucl.ac.be
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