Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia.

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

[1]  J. Kaplan,et al.  CCC1 Suppresses Mitochondrial Damage in the Yeast Model of Friedreich's Ataxia by Limiting Mitochondrial Iron Accumulation* , 2000, The Journal of Biological Chemistry.

[2]  J. Cooper,et al.  Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia. , 2000, Human molecular genetics.

[3]  J. Adamec,et al.  Yeast and Human Frataxin Are Processed to Mature Form in Two Sequential Steps by the Mitochondrial Processing Peptidase* , 1999, The Journal of Biological Chemistry.

[4]  F. Foury.  Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain , 1999, FEBS letters.

[5]  C. Gellera,et al.  The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis. , 1999, Human molecular genetics.

[6]  D. Radisky,et al.  The Yeast Frataxin Homologue Mediates Mitochondrial Iron Efflux , 1999, The Journal of Biological Chemistry.

[7]  M. Koenig,et al.  Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase. , 1998, Human molecular genetics.

[8]  D. Pain,et al.  Mt-Hsp70 Homolog, Ssc2p, Required for Maturation of Yeast Frataxin and Mitochondrial Iron Homeostasis* , 1998, The Journal of Biological Chemistry.

[9]  Douglas C. Wallace,et al.  Radicals r'aging , 1998, Nature Genetics.

[10]  T. Craig,et al.  Metal mediated sterol receptor-DNA complex association and dissociation determined by electrospray ionization mass spectrometry , 1998, Nature Biotechnology.

[11]  B. Guiard,et al.  The ABC transporter Atm1p is required for mitochondrial iron homeostasis , 1997, FEBS letters.

[12]  A. Munnich,et al.  Aconitase and mitochondrial iron–sulphur protein deficiency in Friedreich ataxia , 1997, Nature Genetics.

[13]  P Trouillas,et al.  Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes. , 1997, Human molecular genetics.

[14]  Michel Koenig,et al.  Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin , 1997, Nature Genetics.

[15]  F. Foury,et al.  Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria , 1997, FEBS letters.

[16]  M. Pandolfo,et al.  Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. , 1997, Science.

[17]  P. Harrison,et al.  The ferritins: molecular properties, iron storage function and cellular regulation. , 1996, Biochimica et biophysica acta.

[18]  P. Patel,et al.  Friedreich's Ataxia: Autosomal Recessive Disease Caused by an Intronic GAA Triplet Repeat Expansion , 1996, Science.

[19]  P K Hansma,et al.  Direct observation of enzyme activity with the atomic force microscope. , 1994, Science.

[20]  G von Heijne,et al.  Cleavage-site motifs in mitochondrial targeting peptides. , 1990, Protein engineering.

[21]  M. Kozak,et al.  At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. , 1987, Journal of molecular biology.

[22]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[23]  T. Moyer,et al.  Routine measurement of calcium, magnesium, copper, zinc, and iron in urine and serum by inductively coupled plasma emission spectroscopy. , 1986, Clinical chemistry.

[24]  A. Tangerås.  Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro. , 1985, Biochimica et biophysica acta.

[25]  P. Aisen,et al.  Iron deposition in apoferritin. Evidence for the formation of a mixed valence binuclear iron complex. , 1985, The Journal of biological chemistry.

[26]  B. Lemieux,et al.  The Cardiomyopathy of Friedreich's Ataxia Morphological Observations in 3 Cases , 1980, Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques.

[27]  R. L. Baldwin,et al.  The Theory Of Sedimentation Analysis , 1958 .

[28]  G. Watt,et al.  A kinetic study of iron release from Azotobacter vinelandii bacterial ferritin. , 1996, Journal of inorganic biochemistry.

[29]  J. Kaplan,et al.  Molecular mechanisms of iron uptake in eukaryotes. , 1996, Physiological reviews.

[30]  W. Fenton,et al.  Proteolytic Processing of Mitochondrial Precursor Proteins , 1996 .

[31]  H. Hansma,et al.  Biomolecular imaging with the atomic force microscope. , 1994, Annual review of biophysics and biomolecular structure.

[32]  M. Worwood,et al.  Iron in biochemistry and medicine. , 1974 .