The Yeast Frataxin Homologue (Yfh1) -ABSTRACT-INTRODUCTION
ABSTRACT
The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In yfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in yfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of yfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved 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 showed that yfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in yfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe–S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.
INTRODUCTION
The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Mutations of FRDA associated with decreased frataxin expression are responsible for Friedreich's ataxia, the most common autosomal-recessive neurodegenerative disease of Caucasians (1,2). Both genes code for mitochondrial proteins that are involved in iron homeostasis and cellular respiration (3–8), but their precise roles are unknown. Cardiac tissues from patients with Friedreich's ataxia exhibit iron deposition, deficiencies in many iron–sulphur cluster enzymes and reduced mitochondrial DNA (8,9). In addition, fibroblasts from these patients show hypersensitivity to oxidative stress that can be rescued by treatment with iron chelators (10). A link to haeme biosynthesis has not been uncovered, and blood, bone marrow and red cell development appear to be normal in patient with frataxin deficits. Frataxin is downregulated during erythroid development, suggesting that this protein is not involved in the high-volume iron trafficking that accompanies red cell production in the bone marrow (11). However, activities of haeme enzymes in other tissues of Friedreich's ataxia patients have not been assessed, leaving open the possibility that tissue-specific haeme deficiencies may exist.
Yeast cells lacking Yfh1p mirror many of the phenotypes observed in disease tissues from patients with Friedreich's ataxia. These cells have defective respiration (3,5,12–14), unstable mitochondrial DNA and hypersensitivity to oxidative stress (3–5). The assembly of Fe–S centres is impaired and cytochrome concentrations are low (4,8). Iron uptake is much greater than in wild-type cells, with most of the iron being found in the mitochondria (3,12). The involvement of Yfh1p in the assembly of Fe–S centres has been described in several studies (8,15–18), but no work has been devoted yet to investigate the possible specific involvement of Yfh1p in the synthesis of haeme, a major iron cofactor synthesized into the mitochondria. An impediment to understanding the function of Yfh1p or frataxin has been the complex nature of the cellular phenotypes resulting from depletion or loss of function. Here we reexamine the role of Yfh1p in iron homeostasis with special emphasis on haeme synthesis. We describe a switch from haeme synthesis to zinc protoporphyrin synthesis that occurs in absence of Yfh1p. A highly sensitive fluorimetric method is used to demonstrate this switch. Previous studies have not noted effects of Yfh1 on haeme formation in yeast, and this may be due to the high frequency of suppressor mutations that mask this phenotype. Here we describe the characteristics of such suppressor mutants, and the effects on haeme formation in the absence of Yfh1p.
ABSTRACT
The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In yfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in yfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of yfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved 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 showed that yfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in yfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe–S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.
INTRODUCTION
The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Mutations of FRDA associated with decreased frataxin expression are responsible for Friedreich's ataxia, the most common autosomal-recessive neurodegenerative disease of Caucasians (1,2). Both genes code for mitochondrial proteins that are involved in iron homeostasis and cellular respiration (3–8), but their precise roles are unknown. Cardiac tissues from patients with Friedreich's ataxia exhibit iron deposition, deficiencies in many iron–sulphur cluster enzymes and reduced mitochondrial DNA (8,9). In addition, fibroblasts from these patients show hypersensitivity to oxidative stress that can be rescued by treatment with iron chelators (10). A link to haeme biosynthesis has not been uncovered, and blood, bone marrow and red cell development appear to be normal in patient with frataxin deficits. Frataxin is downregulated during erythroid development, suggesting that this protein is not involved in the high-volume iron trafficking that accompanies red cell production in the bone marrow (11). However, activities of haeme enzymes in other tissues of Friedreich's ataxia patients have not been assessed, leaving open the possibility that tissue-specific haeme deficiencies may exist.
Yeast cells lacking Yfh1p mirror many of the phenotypes observed in disease tissues from patients with Friedreich's ataxia. These cells have defective respiration (3,5,12–14), unstable mitochondrial DNA and hypersensitivity to oxidative stress (3–5). The assembly of Fe–S centres is impaired and cytochrome concentrations are low (4,8). Iron uptake is much greater than in wild-type cells, with most of the iron being found in the mitochondria (3,12). The involvement of Yfh1p in the assembly of Fe–S centres has been described in several studies (8,15–18), but no work has been devoted yet to investigate the possible specific involvement of Yfh1p in the synthesis of haeme, a major iron cofactor synthesized into the mitochondria. An impediment to understanding the function of Yfh1p or frataxin has been the complex nature of the cellular phenotypes resulting from depletion or loss of function. Here we reexamine the role of Yfh1p in iron homeostasis with special emphasis on haeme synthesis. We describe a switch from haeme synthesis to zinc protoporphyrin synthesis that occurs in absence of Yfh1p. A highly sensitive fluorimetric method is used to demonstrate this switch. Previous studies have not noted effects of Yfh1 on haeme formation in yeast, and this may be due to the high frequency of suppressor mutations that mask this phenotype. Here we describe the characteristics of such suppressor mutants, and the effects on haeme formation in the absence of Yfh1p.
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