The Yeast Frataxin Homologue (Yfh1) DISCUSSION
DISCUSSION
The loss-of-function of YFH1 results in striking maldistribution of cellular iron, with depletion of cytoplasmic iron and accumulation of mitochondrial iron. Here we show that mitochondrial iron in yfh1 cells, although present in large excess, is unavailable for haeme synthesis. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, makes primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulates during growth of yfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin is observed in real time. The method for these studies involves direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies show that yfh1 mutant cells exhibit a defect in iron use by ferrochelatase.
Multiple secondary effects and the complex pleiotropic phenotype of these cells has impeded definition of the function of frataxins. The defect in haeme synthesis in Yfh1p depleted cells, however, is readily reversed by reinduction of expression from a regulated promoter, and therefore the phenotype is not due to secondary effects such as mtDNA damage. The precise role of Yfh1p and frataxins in iron delivery for haeme synthesis remains to be defined. A role for Yfh1 in iron solubilization in mitochondria is possible and/or a role in directly handing of iron as described for metallation of copper proteins by copper chaperones (29). Ferrochelatase is able to use soluble ferrous iron as a substrate for iron incorporation into PPIX, and iron is largely present as insoluble ferric particles in yfh1 mitochondria. This could explain why the large excess of iron in these mitochondria is not available for haeme synthesis, and maybe for other iron-requiring processes like Fe–S centre assembly. The involvement of Yfh1p might involve a role in maintaining solubility of iron in mitochondria. Our results show that Hem15p and Yfh1p physically interact in vitro. An in vivo interaction between these two proteins might mediate hand-off of iron for haeme formation. Adamec et al. (30) showed that self-assembled multimers of Yfh1p can sequester more than 3000 atoms of iron, and that iron can be released from the protein shell by a reducing agent. It is tempting to speculate that iron bound to such a Yfh1p intermediate may be the iron donor to ferrochelatase for haeme synthesis. Such a donor should exist as it is unlikely that there is ‘free’ iron in mitochondria, which is an environment particularly sensitive to radical reactions.
The role of Yfh1p in iron delivery for haeme synthesis recalls the recently described role of Yfh1p in iron delivery for Fe–S cluster synthesis in mitochondria (8,15–18). The precise function of Yfh1p is likewise undefined in this process. A recent study by Puccio et al. (17) analysed the sequence of events in tissues of transgenic mice following frataxin depletion. They found that the Fe–S assembly defect appeared prior to mitochondrial iron accumulation, suggesting a primary role for frataxin in iron utilization. Our work also indicates that Yfh1 is involved in mitochondrial iron use (for haeme synthesis as for Fe–S cluster synthesis) and for correct partitioning of iron between mitochondrial and cytoplasmic pools. Definition of the mechanisms of these effects will require additional work, and the identification of additional components of the mitochondrial iron transport system(s).
The cytochrome-deficient phenotype of yfh1 cells is often masked by nuclear mutation(s) that suppress the phenotype. The features of strains carrying such suppressor mutations are striking. Haeme synthesis is restored as indicated by complete recovery of cytochrome spectra and disappearance of Zn-PPIX. Haeme synthesis is restored, although iron accumulation and Fe–S cluster deficiency phenotypes are intermediate between the wild-type and deletion phenotypes. We frequently encountered such genetic changes with various yfh1 strains: strains were cytochrome-deficient when freshly isolated and acquired normal pigmentation after a few culture cycles on complete medium. Our finding that yfh1 cells accumulate suppressor mutation(s) fits well with a recent work (19), showing that the absence of Yfh1 in yeast leads to nuclear damage, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents. The frequency of these events may explain why the link of Yfh1p with cytochromes and haeme synthesis has not previously been described. The implication of the results is that the requirement of Yfh1p in haeme synthesis can be bypassed by nuclear gene mutations. Identifying the suppressor mutation(s) that allow(s) yfh1 cells to recover normal cytochrome synthesis and respiration is being undertaken. The unidentified suppressor mutation(s) might result in solubilizing part of the iron accumulated into yfh1 mitochondria, making it available for haeme synthesis, as suggested by our Mössbauer analysis. The low expression of frataxin in erythroid cells (11) and the lack of red cell phenotypes in the human frataxin deficiency disease Friedreich ataxia (11) likewise suggest that haeme synthesis can occur in the absence of frataxin in some settings.
Ferrochelatases from diverse species as mammals, Drosophila, Schizosaccharomyces pombe and some bacteria possess 2Fe–2S clusters that are critical for activity (reviewed in 31). In view of the role of frataxin in Fe–S cluster formation, an effect of frataxin deficiency on ferrochelatase function in these species might be inferred. By contrast, the ferrochelatase of S. cerevisiae lacks such as Fe–S cluster and appears to be an outlier in this regard. One can speculate about the implications of this difference for the regulatory links between frataxin and ferrochelatase function. Perhaps, the effects of frataxin on Fe–S cluster assembly being insufficient to inactivate ferrochelatase in S. cerevisiae, alternative regulatory mechanisms evolved instead. At least two such mechanisms are observed here. First, a transcriptional mechanism is implied by the decreased Hem15 transcript levels observed in the absence of Yfh1. The mediators of this response are not Aft1 or Aft2, the previously identified iron regulatory proteins (22,23). Second a post-transcriptional effect of Yfh1 is required for active ferrochelatase function. This might involve a role of Yfh1 in delivery of the substrate iron for haeme formation. Alternative possibilities are that Yfh1 activates ferrochelatase activity by producing a folding or conformational change in the protein, or by contribution of a catalytic iron atom that serves a function analogous to the regulatory Fe–S cluster. The direction of these regulatory effects makes sense in terms of coupling haeme synthesis to the availability of the critical iron cofactor. In similar fashion, the first step in haeme biosynthesis, -aminolevulinate synthase in erythroid cells, is positively regulated by iron (32) and ferrochelatase in mammals is also regulated by iron via post-transcriptional mechanisms (33). Many questions remain: the molecular details of the regulatory effects of Yfh1 on ferrochelatase function remain to be defined. Also a question exists as to whether similar control mechanisms will be found in mammalian tissues.
DISCUSSION
The loss-of-function of YFH1 results in striking maldistribution of cellular iron, with depletion of cytoplasmic iron and accumulation of mitochondrial iron. Here we show that mitochondrial iron in yfh1 cells, although present in large excess, is unavailable for haeme synthesis. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, makes primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulates during growth of yfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin is observed in real time. The method for these studies involves direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies show that yfh1 mutant cells exhibit a defect in iron use by ferrochelatase.
Multiple secondary effects and the complex pleiotropic phenotype of these cells has impeded definition of the function of frataxins. The defect in haeme synthesis in Yfh1p depleted cells, however, is readily reversed by reinduction of expression from a regulated promoter, and therefore the phenotype is not due to secondary effects such as mtDNA damage. The precise role of Yfh1p and frataxins in iron delivery for haeme synthesis remains to be defined. A role for Yfh1 in iron solubilization in mitochondria is possible and/or a role in directly handing of iron as described for metallation of copper proteins by copper chaperones (29). Ferrochelatase is able to use soluble ferrous iron as a substrate for iron incorporation into PPIX, and iron is largely present as insoluble ferric particles in yfh1 mitochondria. This could explain why the large excess of iron in these mitochondria is not available for haeme synthesis, and maybe for other iron-requiring processes like Fe–S centre assembly. The involvement of Yfh1p might involve a role in maintaining solubility of iron in mitochondria. Our results show that Hem15p and Yfh1p physically interact in vitro. An in vivo interaction between these two proteins might mediate hand-off of iron for haeme formation. Adamec et al. (30) showed that self-assembled multimers of Yfh1p can sequester more than 3000 atoms of iron, and that iron can be released from the protein shell by a reducing agent. It is tempting to speculate that iron bound to such a Yfh1p intermediate may be the iron donor to ferrochelatase for haeme synthesis. Such a donor should exist as it is unlikely that there is ‘free’ iron in mitochondria, which is an environment particularly sensitive to radical reactions.
The role of Yfh1p in iron delivery for haeme synthesis recalls the recently described role of Yfh1p in iron delivery for Fe–S cluster synthesis in mitochondria (8,15–18). The precise function of Yfh1p is likewise undefined in this process. A recent study by Puccio et al. (17) analysed the sequence of events in tissues of transgenic mice following frataxin depletion. They found that the Fe–S assembly defect appeared prior to mitochondrial iron accumulation, suggesting a primary role for frataxin in iron utilization. Our work also indicates that Yfh1 is involved in mitochondrial iron use (for haeme synthesis as for Fe–S cluster synthesis) and for correct partitioning of iron between mitochondrial and cytoplasmic pools. Definition of the mechanisms of these effects will require additional work, and the identification of additional components of the mitochondrial iron transport system(s).
The cytochrome-deficient phenotype of yfh1 cells is often masked by nuclear mutation(s) that suppress the phenotype. The features of strains carrying such suppressor mutations are striking. Haeme synthesis is restored as indicated by complete recovery of cytochrome spectra and disappearance of Zn-PPIX. Haeme synthesis is restored, although iron accumulation and Fe–S cluster deficiency phenotypes are intermediate between the wild-type and deletion phenotypes. We frequently encountered such genetic changes with various yfh1 strains: strains were cytochrome-deficient when freshly isolated and acquired normal pigmentation after a few culture cycles on complete medium. Our finding that yfh1 cells accumulate suppressor mutation(s) fits well with a recent work (19), showing that the absence of Yfh1 in yeast leads to nuclear damage, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents. The frequency of these events may explain why the link of Yfh1p with cytochromes and haeme synthesis has not previously been described. The implication of the results is that the requirement of Yfh1p in haeme synthesis can be bypassed by nuclear gene mutations. Identifying the suppressor mutation(s) that allow(s) yfh1 cells to recover normal cytochrome synthesis and respiration is being undertaken. The unidentified suppressor mutation(s) might result in solubilizing part of the iron accumulated into yfh1 mitochondria, making it available for haeme synthesis, as suggested by our Mössbauer analysis. The low expression of frataxin in erythroid cells (11) and the lack of red cell phenotypes in the human frataxin deficiency disease Friedreich ataxia (11) likewise suggest that haeme synthesis can occur in the absence of frataxin in some settings.
Ferrochelatases from diverse species as mammals, Drosophila, Schizosaccharomyces pombe and some bacteria possess 2Fe–2S clusters that are critical for activity (reviewed in 31). In view of the role of frataxin in Fe–S cluster formation, an effect of frataxin deficiency on ferrochelatase function in these species might be inferred. By contrast, the ferrochelatase of S. cerevisiae lacks such as Fe–S cluster and appears to be an outlier in this regard. One can speculate about the implications of this difference for the regulatory links between frataxin and ferrochelatase function. Perhaps, the effects of frataxin on Fe–S cluster assembly being insufficient to inactivate ferrochelatase in S. cerevisiae, alternative regulatory mechanisms evolved instead. At least two such mechanisms are observed here. First, a transcriptional mechanism is implied by the decreased Hem15 transcript levels observed in the absence of Yfh1. The mediators of this response are not Aft1 or Aft2, the previously identified iron regulatory proteins (22,23). Second a post-transcriptional effect of Yfh1 is required for active ferrochelatase function. This might involve a role of Yfh1 in delivery of the substrate iron for haeme formation. Alternative possibilities are that Yfh1 activates ferrochelatase activity by producing a folding or conformational change in the protein, or by contribution of a catalytic iron atom that serves a function analogous to the regulatory Fe–S cluster. The direction of these regulatory effects makes sense in terms of coupling haeme synthesis to the availability of the critical iron cofactor. In similar fashion, the first step in haeme biosynthesis, -aminolevulinate synthase in erythroid cells, is positively regulated by iron (32) and ferrochelatase in mammals is also regulated by iron via post-transcriptional mechanisms (33). Many questions remain: the molecular details of the regulatory effects of Yfh1 on ferrochelatase function remain to be defined. Also a question exists as to whether similar control mechanisms will be found in mammalian tissues.
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