Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1)

摘要: TheYFH1 geneistheyeasthomologueofthehumanFRDAgene,whichencodesthefrataxinprotein.Saccharomyces cerevisiae cellslackingtheYFH1 geneshowedverylowcytochromecontent.InDyfh1strains,thelevelofferrochelatase(Hem15p)wasverylow,asaresultoftranscriptionalrepressionofHEM15.However,thelowamountofHem15pwasnotthecauseofhaemedeficiencyinDyfh1 cells.Ferrochelatase,amitochondrialprotein,abletomediateinsertionofironorzincintotheporphyrinprecursor,madeprimarilythezincprotoporphyrinproduct.ZincprotoporphyrininsteadofhaemeaccumulatedduringgrowthofDyfh1mutantcellsand,furthermore,preferentialformationofzincprotoporphyrinwasobservedinrealtime.Themethodforthesestudiesinvolveddirectpresentationofporphyrintomitochondriaandtoferrochelataseofpermeabilizedcellswithintactarchitecture,therebyspecificallytestingtheirondeliveryportionofthehaemebiosyntheticpathway.ThestudiesshowedthatDyfh1 mutantcellsaredefectiveinironusebyferrochelatase.Mo¨ssbauerspectroscopicanalysisshowedthatironwaspresentasamorphousnano-particlesofferricphosphateinDyfh1 mitochondria,whichcouldexplaintheunavailabilityofironforhaemesynthesis.Ahighfrequencyofsuppressormutationswasobserved,andthephenotypeofsuchmutantswascharacterizedbyrestorationofhaemesynthesisintheabsenceofYfh1p.Suppressorstrainsshowedanormalcytochromecontent,normalrespiration,butremaineddefectiveinFe–Sproteinsandstillaccumulatedironintomitochondriaalthoughtoalesserextent.Yfh1pandHem15pwereshowntointeractin vitro byBiacorestudies.OurresultssuggestthatYfh1mediatesironusebyferrochelatase.INTRODUCTIONThe YFH1 gene is the yeast homologue of human FRDAgene, which encodes frataxin protein. Mutations FRDAassociated with decreased expression are responsiblefor Friedreich’s ataxia, most common autosomal-recessiveneurodegenerative disease Caucasians (1,2). Both genescode for mitochondrial proteins that involved in ironhomeostasis and cellular respiration (3–8), but their preciseroles unknown. Cardiac tissues from patients withFriedreich’s ataxia exhibit iron deposition, deficiencies manyiron–sulphur cluster enzymes reduced DNA(8,9). In addition, fibroblasts these showhypersensitivity to oxidative stress can be rescued bytreatment chelators (10). A link haeme biosynthesishas not been uncovered, blood, bone marrow red celldevelopment appear normal patient frataxindeficits. Frataxin downregulated during erythroid develop-ment, suggesting this protein high-volume trafficking accompanies cell production inthe (11). However, activities inother have beenassessed, leaving open possibility tissue-specific haemedeficiencies may exist.Yeast cells lacking Yfh1p mirror many phenotypesobserved Friedreich’sataxia. These defective (3,5,12–14),unstable DNA hypersensitivity