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CCC1 Suppresses Mitochondrial Damage in the Yeast Model of Friedreich's Ataxia by Limiting Mitochondrial Iron Accumulation*

  • Opal S. Chen
    Affiliations
    Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132
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  • Jerry Kaplan
    Correspondence
    To whom correspondence should be addressed. Tel.: 801-581-7427; Fax: 801-581-4517
    Affiliations
    Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132
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  • Author Footnotes
    * This work was supported by NIDDK, National Institutes of Health Grant 52380.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 17, 2000DOI:https://doi.org/10.1074/jbc.275.11.7626
      Deletion of YFH1 inSaccharomyces cerevisiae leads to a loss of respiratory competence due to excessive mitochondrial iron accumulation. A suppressor screen identified a gene, CCC1, that maintained respiratory function in a Δyfh1 yeast strain regardless of extracellular iron concentration. CCC1 expression prevented excessive mitochondrial iron accumulation by limiting mitochondrial iron uptake rather than by increasing mitochondrial iron egress. Expression of CCC1 did not result in sequestration of iron in membranous compartments or cellular iron export.CCC1 expression in wild type cells resulted in increased expression of the high affinity iron transport system composed ofFET3 and FTR1, suggesting that intracellular iron is not sensed by the iron-dependent transcription factor Aft1p. Introduction of AFT1 up, a constitutive allele of the iron transcription factor, AFT1, that also leads to increased high affinity iron transport did not prevent Δyfh1 cells from becoming respiratory-incompetent. Although the mechanism by whichCCC1 expression affects cytosolic iron is not known, the data suggest that excessive mitochondrial iron accumulation only occurs when cytosolic free iron levels are high.
      Friedreich's ataxia is a lethal disorder affecting the nervous system and heart. The disorder is due to a triplet expansion in the first intron of the Frataxin gene that results in decreased levels of Frataxin mRNA (
      • Campuzano V.
      • Montermini L.
      • Lutz Y.
      • Cova L.
      • Hindelang C.
      • Jiralerspong S.
      • Trottier Y.
      • Kish S.J.
      • Faucheux B.
      • Trouillas P.
      • Authier F.J.
      • Durr A.
      • Mandel J.L.
      • Vescovi A.
      • Pandolfo M.
      • Koenig M.
      ), which encodes a mitochondrial protein (
      • Koutnikova H.
      • Campuzano V.
      • Foury F.
      • Dolle P.
      • Cazzalini O.
      • Koenig M.
      ). Insight into the function of Frataxin resulted from studies on theSaccharomyces cerevisiae gene, yeastFrataxin homologue (YFH1), which is an orthologue of mammalian Frataxin. In the absence of Yfh1p, iron accumulates within mitochondria, leading to a loss of respiratory activity through the generation of mitochondrial DNA mutations. It is thought that decreased respiratory activity is a consequence of iron-induced oxygen radicals (
      • Babcock M.
      • de Silva D.
      • Oaks R.
      • Davis-Kaplan S.
      • Jiralerspong S.
      • Montermini L.
      • Pandolfo M.
      • Kaplan J.
      ,
      • Wilson R.B.
      • Roof D.M.
      ). The observation of increased iron deposits in Friedreich's ataxia heart biopsies (
      • Lamarche J.B.
      • Cote M.
      • Lemieux B.
      ) and the finding of increased iron in mitochondria of Friedreich's ataxia fibroblasts (
      • Delatycki M.B.
      • Camakaris J.
      • Brooks H.
      • Evans-Whipp T.
      • Thorburn D.R.
      • Williamson R.
      • Forrest S.M.
      ,
      • Wong A.
      • Yang J.
      • Cavadini P.
      • Gellera C.
      • Lonnerdal B.
      • Taroni F.
      • Cortopassi G.
      ) indicate that the yeast model may be an accurate reflection of the pathophysiology of Friedreich's ataxia.
      We initiated a search for genes that could suppress the respiratory deficit in yeast lacking Yfh1p. Here we report that CCC1,previously characterized as a putative Golgi Ca2+/Mn2+ transporter (
      • Fu D.
      • Beeler T.
      • Dunn T.
      ,
      • Lapinskas P.J.
      • Lin S.J.
      • Culotta V.C.
      ), allowedΔyfh1 cells to maintain respiratory activity by preventing the toxic accumulation of mitochondrial iron. The restriction on mitochondrial iron accumulation resulted from reduced mitochondrial iron uptake rather than increased mitochondrial iron efflux. The observation that respiratory activity as well as leucine biosynthesis was unaffected in Δyfh1 cells overexpressingCCC1 implies that Yfh1p is not significantly involved in the biosynthesis, assembly or export of iron-sulfur clusters. Overexpression of CCC1 in wild type cells activated the iron-dependent transcription factor Aft1p, resulting in an increase in iron uptake and cytosolic iron accumulation. Activation of Aft1p suggests that cells sense that cytosolic iron is low, indicating that much of the cytosolic iron was not bioavailable. This reduced concentration of “free” iron did not affect the growth ofΔyfh1 cells but did prevent excessive mitochondrial iron accumulation. These studies demonstrate that toxic mitochondrial iron levels resulting from the loss of Yfh1p may only occur when cytosolic free iron levels are high.

      DISCUSSION

      A genetic screen revealed that CCC1 is a high copy suppressor of the respiratory deficit of Δyfh1 cells. High copy plasmid expression of CCC1 prevented Δyfh1from becoming petite, even in the presence of high iron concentrations.CCC1 overexpression does not prevent cellular iron uptake but does prevent the increased mitochondrial iron accumulation expected of a Δyfh1 strain. This result confirms previous studies demonstrating that the respiratory defect in Δyfh1 cells is due to excessive accumulation of iron within mitochondria (
      • Radisky D.C.
      • Babcock M.C.
      • Kaplan J.
      ). Heart biopsies from patients with Friedreich's ataxia have shown a selective defect in the activity of mitochondrial iron-sulfur proteins (
      • Rotig A.
      • de Lonlay P.
      • Chretien D.
      • Foury F.
      • Koenig M.
      • Sidi D.
      • Munnich A.
      • Rustin P.
      ). A recent report indicated that in the absence of Yfh1p, even in low iron conditions, there was a decrease in aconitase activity, suggesting that Yfh1p was involved in regulating formation of iron-sulfur clusters (
      • Foury F.
      ). A caveat to those experiments is that aconitase protein levels were not measured. In our strain of yeast, however, deletion of YFH1 does not result in significant loss of iron-sulfur-containing enzyme activities, as determined by biologic function. For the studies reported here, we employedΔyfh1 cells that had a deletion in the LEU2gene. These cells could be converted to leucine prototroph by aLEU2-containing plasmid. Leucine biosynthesis requires the activity of Leu1p, a cytosolic enzyme that relies on iron-sulfur clusters for catalytic activity (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ). Deletion of YFH1 does not confer leucine auxotroph, implying that Yfh1p is not required for the formation of cytosolic iron-sulfur clusters. Respiration also requires the activity of iron-sulfur cluster proteins, andCCC1 can preserve respiratory activity in aΔyfh1 strain. These observations suggest thatYFH1 may not be significantly involved in iron-sulfur cluster formation.
      Overexpression of CCC1 preserves respiratory activity by preventing mitochondrial iron accumulation. We determined that Ccc1p does not accelerate the loss of iron from mitochondria. Thus, Ccc1p prevents or limits iron transported into mitochondria. The limitation on mitochondrial iron accumulation is clearly not absolute, as enough iron enters the mitochondria for the synthesis of heme and iron-sulfur clusters. The mechanism by which Ccc1p limits mitochondrial iron uptake is not clear. Ccc1p was characterized as a Mn2+/Ca2+ transporter, based on genetic studies (
      • Fu D.
      • Beeler T.
      • Dunn T.
      ,
      • Lapinskas P.J.
      • Lin S.J.
      • Culotta V.C.
      ). Examination of the data base reveals homologous genes in plants, bacteria, and the archea. No homologues have been identified in either invertebrates (Caenorhabditis elegans) or vertebrates. It is possible that Ccc1p may transport iron, as most Fe2+ transporters are capable of transporting other divalent transition metals, particularly Mn2+ (
      • Gunshin H.
      • Mackenzie B.
      • Berger U.V.
      • Gunshin Y.
      • Romero M.F.
      • Boron W.F.
      • Nussberger S.
      • Gollan J.L.
      • Hediger M.A.
      ,
      • Korshunova Y.O.
      • Eide D.
      • Clark W.G.
      • Guerinot M.L.
      • Pakrasi H.B.
      ,
      • Supek F.
      • Supekova L.
      • Nelson H.
      • Nelson N.
      ). We have shown that Ccc1p does not lead to iron export from cells or sequestration of iron in membranous compartments. Our results indicate that iron accumulates in cytosol and is not recognized by the iron-sensing transcription factor Aft1p. We do not know the form or species (Fe2+ or Fe3+) of this stored iron. Cells overexpressing Ccc1p perceive an apparent “low” iron content and increase the transcription of the iron regulon, resulting in increased activity of the high affinity iron transport system.
      Increased activity of the high affinity iron transport inCCC1-overexpressing cells is an indication that cells “sense” low iron levels. Expression of the high affinity iron transport system, however, does not necessarily result in iron-limited growth; the activities of a number of essential iron-containing proteins, such as methyl sterol oxidase, Δ9-fatty acid desaturase, are unaffected as shown by robust rates of cell growth. The mechanism by which mitochondria can adjust iron accumulation with respect to cytosolic iron levels is unknown. Restriction of cytosolic “free iron” either by environmental iron restriction (
      • Radisky D.C.
      • Babcock M.C.
      • Kaplan J.
      ,
      • Foury F.
      ), genetic ablation of high affinity iron transport systems (
      • Radisky D.C.
      • Babcock M.C.
      • Kaplan J.
      ), or genetic manipulation of intracellular iron content (CCC1overexpression) prevents excessive mitochondrial iron accumulation in cells lacking YFH1.

      Acknowledgments

      We express our appreciation to our colleagues for help in preparing this manuscript.

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