Frataxin and Mitochondrial Carrier Proteins, Mrs3p and Mrs4p, Cooperate in Providing Iron for Heme Synthesis
Year: 2005
Type: article
Abstract: 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 Δyfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (ΔΔΔ) did not. When wild-type, Δmrs3/4, Δyfh1, and ΔΔΔ 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 ΔΔΔ than in the individual Δmrs3/4 or Δyfh1 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 ΔΔΔ), acting to protect bioavailable iron within mitochondria and to facilitate its use 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 Δyfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (ΔΔΔ) did not. When wild-type, Δmrs3/4, Δyfh1, and ΔΔΔ 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 ΔΔΔ than in the individual Δmrs3/4 or Δyfh1 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 ΔΔΔ), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis. Frataxin is a mitochondrial protein, the deficiency of which has been implicated in the human disease Friedreich's ataxia (1Campuzano V. Montermini L. Molto M.D. Pianese L. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar). This is a progressive disease in which specialized tissues (neurons of dorsal root ganglia, cardiac muscle cells, and pancreatic beta cells) undergo mitochondrial degeneration and cell death (2Puccio H. Simon D. Cossee M. Criqui-Filipe P. Tiziano F. Melki P. M. PubMed Scopus Google Scholar). A to iron has been the of a yeast for the yeast frataxin homolog (YFH1) M. D. Montermini L. M. Science. PubMed Scopus Google Scholar). yeast iron in mitochondria E. A. PubMed Scopus Google the iron proteins, and heme proteins, or E. A. PubMed Scopus Google A. D. A. P. PubMed Scopus Google Scholar). The iron in mitochondria of the phenotypes of the frataxin mutant to be of to proteins, and D. PubMed Scopus Google PubMed Scopus Google Scholar). to the of frataxin be the to of lack of frataxin from Frataxin has a conserved and M. PubMed Google A. PubMed Scopus Google and yeast E. A. Scopus Google been and The a of or and in a with a and a the The of frataxin in of in and phenotypes of formation M. Scopus Google M. Science. PubMed Scopus Google heme E. A. PubMed Scopus Google A. D. A. P. PubMed Scopus Google A. E. PubMed Scopus Google and iron PubMed Scopus Google been of within mitochondria to an role in frataxin and with PubMed Scopus Google M. Science. PubMed Scopus Google and E. A. PubMed Scopus Google PubMed Scopus Google been is a protein an in PubMed Scopus Google PubMed Scopus Google and a role of frataxin in has been M. Scopus Google A. PubMed Scopus Google PubMed Scopus Google Scholar). data also of in in in with frataxin to A. V. F. PubMed Scopus Google A. PubMed Scopus Google Scholar). is the mitochondrial in iron into porphyrin to with a role in heme synthesis, a of frataxin and has been E. A. PubMed Scopus Google E. A. Scopus Google PubMed Scopus Google Scholar). and proteins in the mitochondrial proteins to the mitochondrial carrier and been implicated in iron F. PubMed Scopus Google Scholar). data these carriers to transport iron into mitochondria A. PubMed Scopus Google the mutant phenotypes from deletions of in the Δmrs3/4 mutant strains to which and which L. PubMed Scopus Google Scholar). of the mitochondrial carriers in of iron for heme be to heme has been observed in the Δmrs3/4 of heme synthesis, and these low or in mutants in heme in Scholar). in the Δmrs3/4 mutant were F. PubMed Scopus Google Scholar). a genetic and frataxin and Mrs3/4p in heme synthesis. the yeast strains were in the of oxygen and shifted to a for heme and iron toxicity. The of the Mrs3/4p carriers and frataxin in iron for heme could then be these of the Mrs3/4p carriers were implicated in iron into mitochondria, whereas Yfh1p was implicated in iron bioavailable within and was from growth in the of and for to was from a to Δmrs3/4 was deletions for a and MRS3 into PubMed Scopus Google Scholar). of the was the Δyfh1 was from the Δyfh1 is the and with the M. A. D. PubMed Scopus Google Scholar). The was which is in the of the The mutant of the was was a from in which deletions for a and MRS3 been The was with and the mutant was to ΔΔΔ or were strains of and with a of the strains with the was in and the was from the with Δyfh1 or ΔΔΔ mutants were with the and the was of the was for for the also a was strains of with and in an yeast and of yeast and a for of to which and iron were of iron and were to of iron A. Science. 1996; 271: PubMed Scopus Google Scholar). the were and or were in a of and into a of and then the and to with to a of growth, were in an with a growth of was the in were cultures or with assay was E. M. A. PubMed Scopus Google Scholar). of permeabilized of or mitochondria of were in with these were to of of of was to a was then to a The was the of of from cells, and was for The of was to the H. F. PubMed Scopus Google Scholar). was in permeabilized in assay with or The assay of to time H. PubMed Google Scholar). in was to mitochondrial in in of assay The was with for in a with a A was with and The assay was of iron from a of in an A time was and the in of the the in the PubMed Google PubMed Scopus Google Scholar). were in with and with the cultures were in and in an of an of were in air for an with and then with were with for were with in of for were with and to in of of of with for were then and in The were into and to D. PubMed Scopus Google E. L. PubMed Scopus Google Scholar). in of permeabilized of was and to assay of in a a heme precursor H. P. PubMed Scopus Google was or were to the appearance of and the appearance of in was in and with in a was with a and into the assay to a of was the time the iron and with the permeabilized for or from to were was P. PubMed Google the permeabilized with the iron and from a in and to the of the in with of permeabilized was in a for were in assay and the was to and were and the was incubated for was and the was incubated for an The was the in and then of was The was for was and the was for The was for and of the was and to of cell were previously P. P. Google with an of with of in the heme of the was the Scholar). was the permeabilized to and in to mitochondrial H. D. PubMed Scopus Google Scholar). cell or mitochondrial were the permeabilized or mitochondria to were a protein assay from and Δyfh1 of iron in mitochondria and of iron proteins, has been to in of and of MRS3 and in the Δyfh1 (ΔΔΔ) been to the mitochondrial iron F. PubMed Scopus Google Scholar). in mutant from mitochondrial iron be phenotypes from of iron or iron be A was with to and then to The and mutants in the or for of the and genetic were the Δyfh1 was and in with and in of the mutants, a severe heme with the Δyfh1 was E. A. PubMed Scopus Google Scholar). a the triple mutant ΔΔΔ), a Δyfh1 with was the and phenotypes of or its to heme deficiency been the of F. PubMed Scopus Google Scholar). from strains were in which the Δyfh1 was a a were the and the Δyfh1 the Δmrs3/4 or the ΔΔΔ The were from the strains with the Δyfh1 The which in growth were then and were were and were with The of Δyfh1 and Δyfh1 The of the Δmrs3/4 of all and and were from in all the Δmrs3/4 and Δyfh1, were observed all these were to and strains were in and growth was a was from Δmrs3/4 and time in The Δyfh1 more longer time and time in The ΔΔΔ was the and time in The of the triple mutant was to to the of the growth of the triple mutant to to of mutant of the ΔΔΔ growth were also in of the triple mutant of and the of growth conditions were to the of the severe growth of the ΔΔΔ all the triple and in with and The for is could be to iron different iron of cells, or of alternative and iron under was to with of different Δmrs3/4, Δyfh1, and ΔΔΔ strains were for in growth, and were then or in air were to aerobic the Δmrs3/4, Δyfh1, and The of the is to of which a of the The in the Δyfh1 and ΔΔΔ be to slower growth in these strains The of the and Scholar). The were also with the growth of all strains and the and Δmrs3/4 was of these is the from to aerobic conditions an for heme synthesis, to for under aerobic conditions L. The and of the and Scholar). deficiency was a heme of the from air to growth in the Δyfh1 mutant was more severe than in the or Δmrs3/4, was the growth was severe in and was observed with to the in the of to did to of growth the mutant strains with of and of were for these these heme was of of in synthesis. growth was to the of heme The of and was to the of with the of the Δyfh1 which growth under these conditions was the of the iron to and were to and iron was an iron with different of iron A. Science. 1996; 271: PubMed Scopus Google Scholar). The of strains was and in air with these different iron The growth of the Δmrs3/4 mutant with was in iron The Δmrs3/4 more than in A and or with iron and in with and or and iron. This has been F. PubMed Scopus Google L. PubMed Scopus Google Scholar). the Δyfh1 and triple ΔΔΔ mutants also growth was iron Δyfh1 or ΔΔΔ in and more in air h) to the the of Δmrs3/4 under iron was for the Δyfh1 and for the ΔΔΔ growth was more severe with longer air The of iron were for the ΔΔΔ and more air than h), Δyfh1 and ΔΔΔ strains were and iron of growth was observed with and growth of all mutants heme deficiency a role in growth of these mutants Δyfh1, and and oxygen and iron these and of strains Δmrs3/4, Δyfh1, and ΔΔΔ were for growth of all strains was The cultures were then shifted to air for with the heme deficiency of mutants with the was and was severe in the ΔΔΔ acting porphyrin and iron in the mitochondrial to the of the protein and and previously observed a in protein in Δyfh1 strains in air E. A. PubMed Scopus Google A. PubMed Scopus Google E. PubMed Scopus Google Scholar). This was more in the ΔΔΔ mutant in air Δyfh1 and ΔΔΔ from and to air for were in these mutants with protein was in Δmrs3/4 mutants under The for the in protein in Δyfh1 and ΔΔΔ has been The of a carrier protein of the mitochondrial carrier protein were Yfh1p was in the strains and in in the Δmrs3/4 with protein and protein were from permeabilized from cultures and shifted to air in for proteins were a to and incubated with to protein frataxin or the mitochondrial carrier protein assay for from of permeabilized were in of and to air for The was in a and with to was and a was iron was from an was and under conditions to the permeabilized cell was a assay PubMed Google PubMed Scopus Google in mitochondrial to which iron and were to of protein were from permeabilized growth and in were then with of and the in a The assay was iron and the of for wild-type, Δyfh1, and ΔΔΔ strains were Δmrs3/4 more than the The of of more than of fluorescent to heme in of the the heme deficiency of the mutants the ΔΔΔ be lack of protein or the of in of permeabilized was to be the of were to of mitochondria of This of be to of to the to to heme in the permeabilized of the ΔΔΔ was to deficiency of which was in to lack of iron and of and mitochondria were for of the strains Δmrs3/4, Δyfh1, and cells, there was a in iron in all of the mutants with The mitochondria, iron in the Δyfh1 and in Δmrs3/4 mitochondria, in the ΔΔΔ the cellular iron of the ΔΔΔ mutant was the mitochondrial iron was This of the triple mutant has been F. PubMed Scopus Google L. PubMed Scopus Google Scholar). of the mutants, the iron could for heme synthesis, a is the of the iron in its and were is iron and is also the of heme and E. P. Google E. P. in Scholar). was in the mutants the to air and growth for The was in the and in all mutants, the picture was different growth in the of iron in the was and in Δmrs3/4 was in Δyfh1 and ΔΔΔ were The of these is of the of of and lack of heme in the Δyfh1 and ΔΔΔ strains could for the deficiency of in is a to was in the of strains for in was from in the Δmrs3/4 mutant was in the Δyfh1 mutant and more in the ΔΔΔ with heme deficiency in strains in from was more use of a permeabilized cell of porphyrin to mitochondria E. A. PubMed Scopus Google Scholar). As strains were anaerobically, shifted to air in for The yeast were with to and in in to the D. PubMed Scopus Google Scholar). to the of the were permeabilized cells, mitochondrial were were in was to the mitochondria the the was of the permeabilized to D. PubMed Scopus Google Scholar). from Δmrs3/4, Δyfh1, and ΔΔΔ were presented with a precursor of the of the iron and all strains in with in formation of the the This of heme is in the mutants and in all of the of for the of heme the of heme biosynthesis, the fluorescent is to in the mitochondrial iron is also presented to which porphyrin to heme, a iron to for heme with of the fluorescent to wild-type, Δyfh1, and Δmrs3/4 permeabilized did fluorescent iron in the of in the permeabilized from the ΔΔΔ there was a fluorescent rapid formation of and to to heme The in heme was the of formation in the of the in the of ΔΔΔ in and the was in the triple an alternative of from and was in the of and was in the of in all strains under these the data formation of heme from was in the ΔΔΔ This was a in the genetic of the the defect was a of whereas was in the individual mutants or the individual mutants in in iron for heme synthesis. could be and in the more and of mutants was to and then to in air for an or in were and total heme were the Scholar). of growth in heme were in Δmrs3/4, more in Δyfh1, and in ΔΔΔ than with This of was of growth in the heme deficiency of the Δyfh1 mutant in was more the time than the time heme was in all the strains under the conditions the of the were to for heme formation the in air and the in air The for heme was and to an to the of mitochondrial to aerobic The of total heme did the time for the Δmrs3/4 The of heme formation in the Δyfh1 cells, with time in air to the time the triple the time heme formation was and the was the to time in air growth was a of the total cellular heme and the of its in the different cellular heme Δmrs3/4, Δyfh1, and ΔΔΔ were and shifted to air in for or heme of the was the in in of of in a were low with the oxygen for and in heme biosynthesis, were in for of the strains PubMed Google Scholar). the to cytochromes, and and and could be a was in the and in the more the Δmrs3/4 were and and these were in than in the were also and in the time in the the Δyfh1 were and with A was also and a was the ΔΔΔ a in was the time and defect was more severe than in the more time in the was with total heme heme did under these in of and in were in shifted to air in and permeabilized for an or was to the and iron. these from in in the were in a of was and the of iron were of were with or and data for longer time the appearance and disappearance of fluorescent porphyrin and were also The of formation from in the was the of iron and the for the Δyfh1 mutant were in Δmrs3/4 and ΔΔΔ to iron and the in the time in and in iron was to in these mutants, a slower than in the The formation with or iron was in the ΔΔΔ than in the with the more severe of in permeabilized of time in air and iron. were and were for for the were were with presented with and in and time in and of and an of the with the the is in the with the the is in shifted to air for iron. in iron. Δmrs3/4, in iron. Δyfh1, in iron. in iron. shifted to air for with to iron. in with iron. Δmrs3/4, in with iron. Δyfh1, in with iron. in with iron. shifted to air for iron. wild-type, in iron. Δmrs3/4, in iron. Δyfh1, in iron. in iron. shifted to air for with the of iron. in with iron. Δmrs3/4, in with iron. Δyfh1, in with iron. in with the the of formation in the was iron in the the Δmrs3/4 and ΔΔΔ mutants, the of iron was with the and the of iron in these mutants and were more than of a iron transport different from the The for the Δyfh1 mutant were in the time of in these to be the Δyfh1 with or with the of a iron for heme in these were for the of porphyrin and the appearance and disappearance of fluorescent porphyrins and were were for the wild-type, Δmrs3/4, Δyfh1, and ΔΔΔ of growth in air and of and iron was with the strains of growth in air and the of and iron was to heme or the of the time the of porphyrin formation and were different for the mutants, were the to iron. these were with the time in the and the the was with the the was in whereas the was than the of the the was in formation to a A and and the was with of in This with the the of in under conditions The of iron to the permeabilized or the fluorescent use of iron and of porphyrin to heme and the Δmrs3/4 the of the was than in the and the of the appearance and disappearance of the were and was in and with the The in of iron for heme was more severe the than the the of iron in the disappearance of all and of to heme, the time for was with the and the Δyfh1 a of was the time the time the of formation was and Δyfh1 A and the was the longer time A with with The defect was more severe with time in air the Δyfh1 in with the Δyfh1 in The of iron was to the iron for heme the time the in with the for the Δyfh1 mutant in This was longer the which iron could longer the defect the in with the for the Δyfh1 mutant in the triple the defect in iron use for heme was This was the of the and A defect was also the in the appearance of the or in or The of iron did the the mutant phenotypes of and appearance of the were iron and the of the time all was and to different was also to heme synthesis. The permeabilized mutants anaerobically, shifted to air for and were incubated with and was and for heme was heme was in the A of heme was in the Δmrs3/4 and Δyfh1 A more of heme was from the ΔΔΔ mutant a the Δmrs3/4 and Δyfh1 strains defective in of iron for heme and the ΔΔΔ defect is more The of frataxin in been from of phenotypes of of these phenotypes to of iron in mitochondria H. M. PubMed Scopus Google Scholar). the role of frataxin in the ΔΔΔ which iron in mitochondria, of the phenotypes to iron from to iron The triple mutant has been was the of and in iron F. PubMed Scopus Google Scholar). also observed iron in the triple in observed growth in ΔΔΔ than in Δmrs3/4 or Δyfh1 observed a defect in of heme, which was a in ΔΔΔ than in Δyfh1 or in the genetic the of with of The in heme and which the of the mitochondrial PubMed Google Scholar). was a and in all of the strains was in of be to of heme in these The of has been previously in Scholar). with permeabilized of porphyrin precursor to mitochondria, and in these in of the porphyrin heme was in of the could the in heme in the mutants to a with iron or iron A to the was the use of growth conditions to is PubMed Google and iron under conditions PubMed Scopus Google Scholar). the use of to a heme and iron from which to the evolving mutant phenotypes to aerobic growth The of data presented for Mrs3/4p and Yfh1p in heme of heme from and iron were observed in the triple mutant and were more severe in the triple mutant than in the individual data is to and there were in of the mutant phenotypes to from to aerobic the to and heme synthesis. in the observed the appearance and of cytochromes, whereas formation was with time in was under growth and heme was to aerobic mitochondrial iron was in the permeabilized cell iron was to the mitochondria, formation of the Δmrs3/4 heme were heme proteins and or under the the of appearance of was with in the or in iron was and mitochondrial iron was in to the total iron. the to were A in mutant was a in of iron for heme in the permeabilized cell The formation was all the of iron iron did to the iron did and the was to heme, with a time the and the was severe the time A for is the Mrs3/4p carriers for iron transport into mitochondria for heme and with rapid whereas an alternative slower also The of the with growth with the more with time in The Mrs3/4p also than the alternative has been A. PubMed Scopus Google Scholar). The for which is a iron of has been the Δyfh1 in the phenotypes were with air the time Δyfh1 in heme synthesis. were total heme were was previously E. A. PubMed Scopus Google and was low and permeabilized cell with The formation of iron to is a with for the iron the and in rapid and of the porphyrin to heme, to the the time the and heme in the Δyfh1 The longer the of heme and the permeabilized cell and of fluorescent porphyrin lack of heme synthesis. the of iron could longer the for these be the time with a lack of an iron required to iron in a bioavailable for use the of of which is bioavailable into mitochondria the for the time iron within mitochondria of proteins and also of D. A. PubMed Scopus Google Scholar). The iron then be to the ΔΔΔ iron in mitochondria were was to a role of Yfh1p of iron heme deficiency was more severe than in the Δmrs3/4 or Δyfh1 with total heme than the and more than the This was with of and heme proteins in the triple The was the time and did with time in was the for the triple the of heme formation was of total heme in This also to heme formation from which to and in the permeabilized cell the triple mutant was iron for to a mitochondrial iron transport defect to lack of and an defect iron transport to lack of This in iron for heme to Mrs3/4p the iron or of Yfh1p protect bioavailable iron from to or Yfh1p has been to with PubMed Scopus Google M. Science. PubMed Scopus Google E. A. PubMed Scopus Google E. A. Scopus Google PubMed Scopus Google and iron E. A. Scopus Google A. H. PubMed Scopus Google Scholar). is a protein which PubMed Scopus Google Scholar). is an of the in the mitochondria H. PubMed Google Scholar). is the in iron into porphyrin to heme in Scholar). The of these is under Yfh1p be to protect iron iron or be in of bioavailable to proteins PubMed Scopus Google Scholar). Yfh1p be an and the protein be the protein formation of which the of in Science. PubMed Scopus Google Scholar). A of Yfh1p has been and the of proteins and to different PubMed Scopus Google PubMed Scopus Google Scholar). The of the mutants proteins is mutants deficiency of proteins in low E. PubMed Scopus Google Scholar). The under low or for to the conditions of observed a of Yfh1p for iron use for heme the of iron which did in the ΔΔΔ and was the mitochondrial iron transport in Δmrs3/4 A is bioavailable iron was in the the for Yfh1p frataxin an iron under conditions in which mitochondrial iron low and heme for is of the Δyfh1 mutant the time the of formation was to the heme was observed the time and iron of the of frataxin of conditions and mutant the frataxin of be a of the is the and frataxin in and iron phenotypes the heme A. E. PubMed Scopus Google Scholar). Frataxin with has been to a of the frataxin E. A. Scopus Google and iron for heme be of the whereas be frataxin The frataxin and conserved and of frataxin also A role of frataxin in formation in yeast and human mitochondria is of frataxin and in yeast PubMed Scopus Google and PubMed Scopus Google Scholar). is and the of and frataxin has been in yeast E. A. PubMed Scopus Google E. A. Scopus Google and PubMed Scopus Google Scholar). there be a role of frataxin in heme in heme protein been observed in Friedreich's ataxia This in frataxin heme to frataxin been observed of or cellular with heme or severe A role for frataxin in heme in also be under conditions M. M. PubMed Scopus Google Scholar). be required to the of yeast or human in iron for heme synthesis. for the and for use of the
Source: Journal of Biological Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Award: G1170948855
OpenAlex ID: W2107386134