Mutation in the Fe-S scaffold protein Isu bypasses frataxin deletion

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe-S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe-S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe-S cluster enzyme activities were improved. The efficiency of new Fe-S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe-S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe-S cluster assembly and/or indirect effects on mitochondrial iron availability.     

Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo

The neurodegenerative disease Friedreich's ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron-storage protein. We identified a mutant Yfh1 defective in iron-induced oligomerization. This mutant protein was able to replace functionally wild-type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization-deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization-induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization-deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron-sulphur cluster biogenesis.     

Partial conservation of functions between eukaryotic frataxin and the Escherichia coli frataxin homolog CyaY

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

Assembly of the iron-binding protein frataxin in Saccharomyces cerevisiae responds to dynamic changes in mitochondrial iron influx and stress level

Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to > or = 24-mer, independent of interactions between Yfh1 and its major iron-binding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.     

Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites

CyaY is the bacterial ortholog of frataxin, a small mitochondrial iron binding protein thought to be involved in iron sulphur cluster formation. Loss of frataxin function leads to the neurodegenerative disorder Friedreich's ataxia. We have solved the solution structure of CyaY and used the structural information to map iron binding onto the protein surface. Comparison of the behavior of wild-type CyaY with that of a mutant indicates that specific binding with a defined stoichiometry does not require aggregation and that the main binding site, which hosts both Fe(2+) and Fe(3+), occupies a highly anionic surface of the molecule. This function is conserved across species since the corresponding region of human frataxin is also able to bind iron, albeit with weaker affinity. The presence of secondary binding sites on CyaY, but not on frataxin, hints at a possible polymerization mechanism. We suggest mutations that may provide further insights into the frataxin function.     

Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain.

Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects. We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild-type and YFH1 deletant strains. Under these conditions, the activity of the respiratory complexes is restored. However, the activity of the mitochondrial aconitase, a 4Fe-4S cluster-containing protein, remains low. The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur. Yfh1p might control the synthesis of iron-sulfur clusters in mitochondria.     

Conformational stability of human frataxin and effect of Friedreich's ataxia-related mutations on protein folding

The neurodegenerative disorder FRDA (Friedreich's ataxia) results from a deficiency in frataxin, a putative iron chaperone, and is due to the presence of a high number of GAA repeats in the coding regions of both alleles of the frataxin gene, which impair protein expression. However, some FRDA patients are heterozygous for this triplet expansion and contain a deleterious point mutation on the other allele. In the present study, we investigated whether two particular FRDA-associated frataxin mutants, I154F and W155R, result in unfolded protein as a consequence of a severe structural modification. A detailed comparison of the conformational properties of the wild-type and mutant proteins combining biophysical and biochemical methodologies was undertaken. We show that the FRDA mutants retain the native fold under physiological conditions, but are differentially destabilized as reflected both by their reduced thermodynamic stability and a higher tendency towards proteolytic digestion. The I154F mutant has the strongest effect on fold stability as expected from the fact that the mutated residue contributes to the hydrophobic core formation. Functionally, the iron-binding properties of the mutant frataxins are found to be partly impaired. The apparently paradoxical situation of having clinically aggressive frataxin variants which are folded and are only significantly less stable than the wild-type form in a given adverse physiological stress condition is discussed and contextualized in terms of a mechanism determining the pathology of FRDA heterozygous.     

Oxidative stress promotes degradation of the Irr protein to regulate haem biosynthesis in Bradyrhizobium japonicum

The haem proteins catalase and peroxidase are stress response proteins that detoxify reactive oxygen species. In the bacterium Bradyrhizobium japonicum, expression of the gene encoding the haem biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) is normally repressed by the Irr protein in iron-limited cells. Irr degrades in the presence of iron, which requires haem binding to the protein. Here, we found that ALAD levels were elevated in iron-limited cells of a catalase-deficient mutant, which corresponded with aberrantly low levels of Irr. Irr was undetectable in wild-type cells within 90 min after exposure to exogenous H2O2, but not in a haem-deficient mutant strain. In addition, Irr did not degrade in response to iron in the absence of O2. The findings indicate that reactive oxygen species promote Irr turnover mediated by haem, and are involved in iron-dependent degradation. We demonstrated Irr oxidation in vitro, which required haem, O2 and a reductant. A truncated Irr mutant unable to bind ferrous haem does not degrade in vivo, and was not oxidized in vitro. We suggest that Irr oxidation is a signal for its degradation, and that cells sense and respond to oxidative stress through Irr to regulate haem biosynthesis.     

Apo forms of cytochrome C550 and cytochrome cd1 are translocated to the periplasm of Paracoccus denitrificans in the absence of haem incorporation caused by either mutation or inhibition of haem synthesis

An apo form of cytochrome C₅₅₀ can be detected by immunoblotting cell-free extracts of a mutant of Paracoccus denitrificans that is deficient in c-type cytochromes. This apoprotein is found predominantly in the periplasm, the location of the holocytochrome in the wild-type organism, indicating that translocation of the polypeptide occurs in the absence of haem attachment. The polypeptide molecular weight, as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, is indistinguishable from that of the holoprotein and the chemically prepared apoprotein; this suggests that the N-terminal signal sequence is removed in the mutant as in the wild-type organism. In the presence of levulinic acid, an inhibitor of haem biosynthesis, apocytochrome c₅₅₀ and aponitrite reductase (cytochrome cd₁) accumulated in the periplasm of wild-type cells. Synthesis of these apoproteins was blocked by chloramphenicol. Thus in P. denitrificans the synthesis of these polypeptides is neither autoregulated nor regulated by the availability of haem. That the apoproteins appear in the periplasm argues against the possibility of polypeptide/haem co-transport from cytoplasm to periplasm. These observations are related to, and contrasted with, the biosynthesis of c-type cytochromes in eukaryotic cells.