The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins

The mitochondrial matrix protein frataxin is depleted in patients with Friedreich's ataxia, the most common autosomal recessive ataxia. While frataxin is important for intracellular iron homeostasis, its exact cellular role is unknown. Deletion of the yeast frataxin homolog YFH1 yields mutants ((Delta)yfh1) that, depending on the genetic background, display various degrees of phenotypic defects. This renders it difficult to distinguish primary (early) from secondary (late) consequences of Yfh1p deficiency. We have constructed a yeast strain (Gal-YFH1) that carries the YFH1 gene under the control of a galactose-regulated promoter. Yfh1p-deficient Gal-YFH1 cells are far less sensitive to oxidative stress than (Delta)yfh1 mutants, maintain mitochondrial DNA, and synthesize heme at wild-type rates. Yfh1p depletion causes a strong reduction in the assembly of mitochondrial Fe/S proteins both in vivo and in detergent extracts of mitochondria. Impaired Fe/S protein biogenesis explains the respiratory deficiency of Gal-YFH1 cells. Furthermore, Yfh1p-depleted Gal-YFH1 cells show decreased maturation of cytosolic Fe/S proteins and accumulation of mitochondrial iron. This latter phenotype is common for defects in cytosolic Fe/S protein assembly. Together, our data demonstrate a specific role of frataxin in the biosynthesis of cellular Fe/S proteins and exclude most of the previously suggested functions. Friedreich's ataxia may therefore represent a disorder caused by defects in Fe/S protein maturation.     

Mitochondrial intermediate peptidase and the yeast frataxin homolog together maintain mitochondrial iron homeostasis in Saccharomyces cerevisiae.

Friedreich's ataxia (FRDA) is a neurodegenerative disease typically caused by a deficiency of frataxin, a mitochondrial protein of unknown function. In Saccharomyces cerevisiae, lack of the yeast frataxin homolog ( YFH1 gene, Yfh1p polypeptide) results in mitochondrial iron accumulation, suggesting that frataxin is required for mitochondrial iron homeostasis and that FRDA results from oxidative damage secondary to mitochondrial iron overload. This hypothesis implies that the effects of frataxin deficiency could be influenced by other proteins involved in mitochondrial iron usage. We show that Yfh1p interacts functionally with yeast mitochondrial intermediate peptidase ( OCT1 gene, YMIP polypeptide), a metalloprotease required for maturation of ferrochelatase and other iron-utilizing proteins. YMIP is activated by ferrous iron in vitro and loss of YMIP activity leads to mitochondrial iron depletion, suggesting that YMIP is part of a feedback loop in which iron stimulates maturation of YMIP substrates and this in turn promotes mitochondrial iron uptake. Accordingly, YMIP is active and promotes mitochondrial iron accumulation in a mutant lacking Yfh1p ( yfh1 [Delta]), while genetic inactivation of YMIP in this mutant ( yfh1 [Delta] oct1 [Delta]) leads to a 2-fold reduction in mitochondrial iron levels. Moreover, overexpression of Yfh1p restores mitochondrial iron homeostasis and YMIP activity in a conditional oct1 ts mutant, but does not affect iron levels in a mutant completely lacking YMIP ( oct1 [Delta]). Thus, we propose that Yfh1p maintains mitochondrial iron homeostasis both directly, by promoting iron export, and indirectly, by regulating iron levels and therefore YMIP activity, which promotes mitochondrial iron uptake. This suggests that human MIP may contribute to the functional effects of frataxin deficiency and the clinical manifestations of FRDA.     

Mitochondrial frataxin interacts with ISD11 of the NFS1/ISCU complex and multiple mitochondrial chaperones

The neurodegenerative disorder Friedreich's ataxia (FRDA) is caused by mutations in frataxin, a mitochondrial protein whose function remains controversial. Using co-immunoprecipitation and mass spectrometry we identified multiple interactors of mitochondrial frataxin in mammalian cells. One interactor was mortalin/GRP75, a homolog of the yeast ssq1 chaperone that integrates iron-sulfur clusters into imported mitochondrial proteins. Another interactor was ISD11, recently identified as a component of the eukaryotic complex Nfs1/ISCU, an essential component of iron-sulfur cluster biogenesis. Interactions between frataxin and ISD11, and frataxin and GRP75 were confirmed by co-immunoprecipitation experiments in both directions. Immunofluorescence analysis demonstrated that ISD11 co-localized with both frataxin and with mitochondria. The point mutations I154F and W155R in frataxin cause FRDA and are clustered to one surface of the protein, and these mutations decrease the interaction of frataxin with ISD11. The frataxin/ISD11 interaction was also decreased by the chelator EDTA, and was increased by supplementation with nickel but not other metal ions. Nickel supplementation rescued the defective interaction of mutant frataxin I154F and W155R with ISD11. Upon ISD11 depletion by siRNA in HEK293T cells, the amount of the Nfs1/ISCU protein complex declined, as did the activity of the iron-sulfur cluster enzyme aconitase, while the cellular iron content was increased, as seen in tissues from FRDA patients. Furthermore, ISD11 mRNA levels were decreased in FRDA patient cells. These data suggest that frataxin binds the iron-sulfur biogenesis Nfs1/ISCU complex through ISD11, that the interaction is nickel-dependent, and that multiple consequences of frataxin deficiency are duplicated by ISD11 deficiency.     

Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae

Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes. Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease. Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity. Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown. We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p. We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively. The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast. The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency. Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype. These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.     

Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of frataxin, a conserved mitochondrial protein of unknown function. Mitochondrial iron accumulation, loss of iron-sulfur cluster-containing enzymes and increased oxidative damage occur in yeast and mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA patients, suggesting that frataxin may be involved in export of iron from the mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative damage. We have previously shown that yeast frataxin has structural and functional features of an iron storage protein. In this study we have investigated the function of human frataxin in Escherichia coli and Saccharomyces cerevisiae. When expressed in E.coli, the mature form of human frataxin assembles into a stable homopolymer that can bind approximately 10 atoms of iron per molecule of frataxin. The iron-loaded homopolymer can be detected on non-denaturing gels by either protein or iron staining demonstrating a stable association between frataxin and iron. As analyzed by gel filtration and electron microscopy, the homopolymer consists of globular particles of approximately 1 MDa and ordered rod-shaped polymers of these particles that accumulate small electron-dense cores. When the human frataxin precursor is expressed in S.cerevisiae, the mitochondrially generated mature form is separated by gel filtration into monomer and a high molecular weight pool of >600 kDa. A high molecular weight pool of frataxin is also present in mouse heart indicating that frataxin can assemble under native conditions. In radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation with approximately five atoms of (55)Fe bound per molecule. These findings suggest that FRDA results from decreased mitochondrial iron storage due to frataxin deficiency which may impair iron metabolism, promote oxidative damage and lead to progressive iron accumulation.     

Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia

Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.     

Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase

Frataxin is a mitochondrial protein deficient in Friedreich ataxia (FRDA) and which is associated with abnormal intramitochondrial iron handling. We identified the mitochondrial processing peptidase beta (MPPbeta) as a frataxin protein partner using the yeast two-hybrid assay. In in vitro assays, MPPbeta binds frataxin which is cleaved by the reconstituted MPP heterodimer. MPP cleavage of frataxin results in an intermediate form (amino acids 41-210) that is processed further to the mature form. In vitro and in vivo experiments suggest that two C- terminal missense mutations found in FRDA patients modulate interaction with MPPbeta, resulting in a slower maturation process at the normal cleavage site. The slower processing rate of frataxin carrying such missense mutations may therefore contribute to frataxin deficiency, in addition to an impairment of its function.      

Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia

Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by expansion of a trinucleotide repeat within the first intron of the gene that encodes frataxin. In our study, we investigated the regulation of frataxin expression by iron and demonstrated that frataxin mRNA levels decrease significantly in multiple human cell lines treated with the iron chelator, desferal (DFO). In addition, frataxin mRNA and protein levels decrease in fibroblast and lymphoblast cells derived from both normal controls and from patients with FA when treated with DFO. Lymphoblasts and fibroblasts of FA patients have evidence of cytosolic iron depletion, as indicated by increased levels of iron regulatory protein 2 (IRP2) and/or increased IRE-binding activity of IRP1. We postulate that this inferred cytosolic iron depletion occurs as frataxin-deficient cells overload their mitochondria with iron, a downstream regulatory effect that has been observed previously when mitochondrial iron-sulfur cluster assembly is disrupted. The mitochondrial iron overload and presumed cytosolic iron depletion potentially further compromise function in frataxin-deficient cells by decreasing frataxin expression. Thus, our results imply that therapeutic efforts should focus on an approach that combines iron removal from mitochondria with a treatment that increases cytosolic iron levels to maximize residual frataxin expression in FA patients.     

Friedreich's ataxia: from disease mechanisms to therapeutic interventions

Friedreich's ataxia (FRDA) is the most common inherited ataxia. FRDA is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein that is severely reduced in FRDApatients. The function of frataxin has not been established yet. Studies of the yeast and animal model of the disease as well as of tissues from FRDA patients have demonstrated that deficit of frataxin is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of tissue energy metabolism. Pilot studies have shown the potential effect of antioxidant therapy in this condition and provide a strong rationale for designing larger clinical randomized trials.     

The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease. Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency. These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients. FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls. The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40%. Treatment of cells with the intracellular Ca2+chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death. Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine- induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts. These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.      

Friedreich ataxia: the oxidative stress paradox

Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe-S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.     

Reduction in frataxin causes progressive accumulation of mitochondrial damage

Frataxin protein controls iron availability in mitochondria and reduced levels lead to the human disease, Friedreich's ataxia (FRDA). The molecular aspects of disease progression are not well understood. We developed a highly regulatable promoter system for expressing frataxin in yeast to address the consequences of chronically reduced amounts of this protein. Shutting off the promoter resulted in changes normally associated with loss of frataxin including iron accumulation within the mitochondria and the induction of mitochondrial petite mutants. While there was considerable oxidative damage to mitochondrial proteins, the petites were likely due to accumulation of mitochondrial DNA lesions and subsequent DNA loss. Chronically reduced frataxin levels resulted in similar response patterns. Furthermore, nuclear DNA damage was detected in a rad52 mutant, deficient in double-strand break repair. We conclude that reduced frataxin levels, which is more representative of the disease state, results in considerable oxidative damage in both mitochondrial and nuclear DNA.     

Sensitivity of FRDA lymphoblasts to salts of transition metal ions

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease resulting from decreased expression of the nuclear-encoded mitochondrial protein, frataxin. FRDA patients have characteristic iron deposits and dysfunction of mitochondrial enzymes in the heart. Inactivation of the frataxin homologue in yeast causes dysregulation of both mitochondrial iron levels and iron export. Previously, we have observed sensitivity of FRDA fibroblasts to FeCl3 and hydrogen peroxide, results consistent with the hypothesis that FRDA cells may experience increased Fenton chemistry. To determine whether the sensitivity of FRDA cells to transition metal ions is a general or specific property, we have compared the sensitivity of lymphoblasts from FRDA patients and healthy controls to the transition metal salts CoCl2, CuSO4 FeCl3 FeSO4, MnCl2, and ZnCl2. FRDA lymphoblasts were significantly more sensitive to FeCl3 and MnCl2 than control cells. However, there were no significant differences observed in sensitivity to CoCl2, CuSO4, FeSO4 and ZnCl2 in the concentration ranges studied. Thus, the sensitivity of FRDA lymphoblasts exposed to transition metals appears to be specific, and could be relevant to the pathophysiological mechanism, which is discussed.     

Frataxin expression rescues mitochondrial dysfunctions in FRDA cells

Friedreich's ataxia (FRDA) is the result of mutations in the nuclear-encoded frataxin gene, which is expressed in mitochondria. Several lines of evidence have suggested that frataxin is involved in mitochondrial iron homeostasis. We have transfected the frataxin gene into lymphoblasts of FRDA compound heterozygotes (FRDA-CH) with deficient frataxin expression to produce FRDA-CH-t cells in which message and protein are rescued to near-physiological levels. FRDA-CH cells were more sensitive to oxidative stress by challenge with free iron, hydrogen peroxide and the combination, consistent with a Fenton chemical mechanism of pathophysiology, and this sensitivity was rescued to control levels in FRDA-CH-t cells. Iron challenge caused increased mitochondrial iron levels in FRDA-CH cells, and a decreased mitochondrial membrane potential (MMP), both of which were rescued in FRDA-CH-t cells. The rescue of the low MMP, and high mitochondrial iron concentration by frataxin overexpression suggests that these cellular phenotypes are relevant to the central pathophysiological process in FRDA which is aggravated by exposure to free iron. However, even at physiological iron concentrations, FRDA-CH cells had decreased MMP as well as lower activities of aconitase and ICDH (two enzymes supporting MMP), and twice the level of filtrable mitochondrial iron (but no increase in total mitochondrial iron), and the observed phenotypes were either fully or partially rescued in FRDA-CH-t cells. Free iron is known to be toxic. The observation that frataxin deficiency (either directly or indirectly) causes an increase in filtrable mitochondrial iron provides a new hypothesis for the mechanism of cell death in this disease, and could be a target for therapy.     

A structural approach to understanding the iron-binding properties of phylogenetically different frataxins

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin--as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms iron-promoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the beta sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.     

Idebenone delays the onset of cardiac functional alteration without correction of Fe-S enzymes deficit in a mouse model for Friedreich ataxia

Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associated with cardiomyopathy, is caused by severely reduced frataxin, a mitochondrial protein involved in Fe-S cluster assembly. We have recently generated mouse models that reproduce important progressive pathological and biochemical features of the human disease. Our frataxin-deficient mouse models initially demonstrate time-dependent intramitochondrial iron accumulation, which occurs after onset of the pathology and after inactivation of the Fe-S dependent enzymes. Here, we report a more detailed pathophysiological characterization of our mouse model with isolated cardiac disease by echocardiographic, biochemical and histological studies and its use for placebo-controlled therapeutic trial with Idebenone. The Fe-S enzyme deficiency occurs at 4 weeks of age, prior to cardiac dilatation and concomitant development of left ventricular hypertrophy, while the mitochondrial iron accumulation occurs at a terminal stage. From 7 weeks onward, Fe-S enzyme activities are strongly decreased and are associated with lower levels of oxidative stress markers, as a consequence of reduced respiratory chain activity. Furthermore, we demonstrate that the antioxidant Idebenone delays the cardiac disease onset, progression and death of frataxin deficient animals by 1 week, but does not correct the Fe-S enzyme deficiency. Our results support the view that frataxin is a necessary, albeit non-essential, component of the Fe-S cluster biogenesis, and indicate that Idebenone acts downstream of the primary Fe-S enzyme deficit. Furthermore, our results demonstrate that Idebenone is cardioprotective even in the context of a complete lack of frataxin, which further supports its utilization for the treatment of FRDA.     

HSC20 interacts with frataxin and is involved in iron-sulfur cluster biogenesis and iron homeostasis

Friedreich's ataxia is a neurodegenerative disorder caused by mutations in the frataxin gene that produces a predominantly mitochondrial protein whose primary function appears to be mitochondrial iron-sulfur cluster (ISC) biosynthesis. Previously we demonstrated that frataxin interacts with multiple components of the mammalian ISC assembly machinery. Here we demonstrate that frataxin interacts with the mammalian mitochondrial chaperone HSC20. We show that this interaction is iron-dependent. We also show that like frataxin, HSC20 interacts with multiple proteins involved in ISC biogenesis including the ISCU/Nfs1 ISC biogenesis complex and the GRP75 ISC chaperone. Furthermore, knockdown of HSC20 caused functional defects in activity of mitochondrial ISC-containing enzymes and also defects in ISC protein expression. Alterations up or down of frataxin expression caused compensatory changes in HSC20 expression inversely, as expected of two cooperating proteins operating in the same pathway and suggesting a potential therapeutic strategy for the disease. Knockdown of HSC20 altered cytosolic and mitochondrial iron pools and increased the expression of transferrin receptor 1 and iron regulatory protein 2 consistent with decreased iron bioavailability. These results indicate that HSC20 interacts with frataxin structurally and functionally and is important for ISC biogenesis and iron homeostasis in mammals. Furthermore, they suggest that HSC20 may act late in the ISC pathway as a chaperone in ISC delivery to apoproteins and that HSC20 should be included in multi-protein complex studies of mammalian ISC biogenesis.     

The in vivo mitochondrial two-step maturation of human frataxin

Deficiency in the nuclear-encoded mitochondrial protein frataxin causes Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associating spinocerebellar ataxia and cardiomyopathy. Although the exact function of frataxin is still a matter of debate, it is widely accepted that frataxin is a mitochondrial iron chaperone involved in iron-sulfur cluster and heme biosynthesis. Frataxin is synthesized as a precursor polypeptide, directed to the mitochondrial matrix where it is proteolytically cleaved by the mitochondrial processing peptidase to the mature form via a processing intermediate. The mature form was initially reported to be encoded by amino acids 56-210 (m(56)-FXN). However, two independent reports have challenged these studies describing two different forms encoded by amino acids 78-210 (m(78)-FXN) and 81-210 (m(81)-FXN). Here, we provide evidence that mature human frataxin corresponds to m(81)-FXN, and can rescue the lethal phenotype of fibroblasts completely deleted for frataxin. Furthermore, our data demonstrate that the migration profile of frataxin depends on the experimental conditions, a behavior which most likely contributed to the confusion concerning the endogenous mature frataxin. Interestingly, we show that m(56)-FXN and m(78)-FXN can be generated when the normal maturation process of frataxin is impaired, although the physiological relevance is not clear. Furthermore, we determine that the d-FXN form, previously reported to be a degradation product, corresponds to m(78)-FXN. Finally, we demonstrate that all frataxin isoforms are generated and localized within the mitochondria. The clear identification of the N-terminus of mature FXN is an important step for designing therapeutic approaches for FRDA based on frataxin replacement.     

Targeted disruption of hepatic frataxin expression causes impaired mitochondrial function, decreased life span and tumor growth in mice

We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals.     

Maturation of frataxin within mammalian and yeast mitochondria: one-step processing by matrix processing peptidase.

Friedreich's ataxia is a neurodegenerative disease caused by mutations in the nuclear gene encoding frataxin (FRDA). FRDA is synthesized with an N-terminal signal sequence, which is removed after import into mitochondria. We have shown that FRDA was imported efficiently into isolated mammalian or yeast mitochondria. In both cases, the processing cleavage that removed the N-terminal signal sequence occurred in a single step on import, generating mature products of identical mobility. The processing cleavage could be reconstituted by incubating the FRDA preprotein with rat or yeast matrix processing peptidase (MPP) expressed in Escherichia coli. We used these assays to evaluate the import and processing of an altered form of FRDA containing the disease-causing I154F mutation. No effects on import or maturation of this mutated FRDA were observed. Likewise, no effects were observed on import and maturation of the yeast frataxin homolog (Yfh1p) carrying a homologous I130F mutation. These results argue against the possibility that the I154F mutation interferes with FRDA function via effects on maturation. Other mutations can be screened for effects on FRDA biogenesis as described here, by evaluating import into isolated mitochondria and by testing maturation with purified MPP.