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Human Molecular Genetics Advance Access originally published online on July 28, 2004
Human Molecular Genetics 2004 13(19):2279-2288; doi:10.1093/hmg/ddh232
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Human Molecular Genetics, Vol. 13, No. 19 © Oxford University Press 2004; all rights reserved

The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast

Alessandro Campanella1, Grazia Isaya2, Heather A. O'Neill2, Paolo Santambrogio1, Anna Cozzi1, Paolo Arosio3 and Sonia Levi1,*

1Department of Biological and Technological Research, IRCCS H. San Raffaele, Via Olgettina 58, Milano, 20132 Italy, 2Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA and 3Dipartimento Materno Infantile e Tecnologie Biomediche, University of Brescia, 25125 Italy

Received May 12, 2004; Accepted July 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitochondrial ferritin (MtF) is structurally and functionally similar to the cytosolic ferritins, molecules designed to store and detoxify cellular iron. MtF expression in human and mouse is restricted to the testis and few tissues, and it is abundant in the erythroblasts of patients with sideroblastic anemia, where it is thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast mitochondria and processed to functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the respiratory deficiency caused by the loss of frataxin protecting the activity of iron–sulfur enzymes and enabling frataxin-deficient cells to grow on non-fermentable carbon sources. Furthermore, MtF expression prevented the development of mitochondrial iron overload, preserved mitochondrial DNA integrity and increased cell resistance to H2O2. The data show that MtF can substitute for most frataxin functions in yeast, suggesting that frataxin is directly involved in mitochondrial iron-binding and detoxification.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Iron is needed for the synthesis of enzymes essential for respiration, redox reactions and DNA synthesis, and is also potentially toxic for its capacity to catalyze free radical formation (1). Consequently iron homeostasis must be tightly controlled by specific mechanisms. The ones so far characterized are located at the cytoplasmic level and involve the iron regulatory proteins that sense iron levels, and the ferritins (2). These are 24-mer proteins composed of the H- and L-subunit types that sequester excess iron in their large cavity in a bioavailable and non-toxic form (1,3). However, much iron has to enter the mitochondria to be incorporated into heme and Fe/S complexes for the synthesis of enzymes (4,5), but little is known about the regulation of this trafficking and how iron toxicity is prevented. Mitochondrial ferritin (MtF) and frataxin are candidates to play important roles in the regulation of mitochondrial iron homeostasis, although their functional roles have not been fully elucidated. MtF has been recently identified in humans, primates and rodents, and shown to be encoded by an intronless gene, which in human is localized on chromosome 5q23.1 (6,7). It is expressed as a precursor with a long N-terminal extension (approximately 60 residues) that directs mitochondrial targeting. The amino acid sequence of the mature protein fully overlaps that of H-ferritin with 77% identity, including all the key residues for ferroxidase activity, and the study of the human and mouse recombinant proteins confirmed that they have iron-binding capacity and ferroxidase activity comparable to that of the well-characterized cytosolic H-ferritin (7). In addition, its crystallographic structure is remarkably similar to that of the H-ferritin (8). When the MtF precursor was expressed in HeLa cells it was found to be fully processed to the mature 21 kDa protein and to accumulate specifically inside the mitochondria. There it assembled in functional 24-mer ferritin molecules, which were active in taking up iron. This activity, which was linked to the integrity of the ferroxidase center, had a profound effect on cellular iron homeostasis, since it reduced both cytosolic ferritin levels and iron availability (9). It was concluded that MtF has a function similar to that of the well-characterized cytosolic ferritin, differing for the mitochondrial localization and for being composed of a single subunit type. MtFs lack the regulatory IRE sequence, and their expression does not seem to be iron-regulated at the post-transcriptional level (6). In addition, and at variance with the ubiquitous cytosolic ferritins, MtF is expressed in a limited number of tissues, mainly in the testis and spermatocytes (10). Interestingly, high levels of MtF protein have been found in ringed sideroblasts of patients with sideroblastic anemia (11). The mitochondria of these cells contain large iron deposits. Mutations in the ALAS2 gene are responsible for the genetic X-linked form of the disorder (12), whereas unknown factors, possibly linked to mitochondrial defects, are implicated in the sporadic forms (13). The excess iron is sequestered inside the MtF, and, because the sideroblasts live and proliferate, it has been suggested that MtF protects mitochondria from the toxicity of local iron excess (11). This hypothesis is supported by preliminary analysis of transfectant cells (10). Normal erythroblasts do not express detectable MtF, indicating that the protein is induced in the disorder.

Frataxin deficiency is associated with Friedreich ataxia (FRDA), the most common genetic form of ataxia (reviewed in 14,15). It is a mitochondrial protein found in all eukaryotes including yeast. The mature protein is a monomer of ~14 kDa, and its 3D structure does not show evident metal binding sites (16,17). However, it binds iron in vitro (18–21). It was shown that the yeast frataxin, Yfh1p, is activated by Fe(II) in the presence of O2 to form trimers that catalyze iron oxidation (22). Higher iron concentrations induce a stepwise assembly of the protein to higher oligomers that can sequester more than 2000 Fe atoms in ferrihydrite-like polynuclear structures, similar to those found in ferritins (23). In addition, Yfh1p oligomers can bind Fe(II) which is available to ferrochelatase for heme synthesis (21). In fact, a physical interaction between Yfh1p and ferrochelatase has been demonstrated in Biacore experiments (24). Other reports indicated that human frataxin can bind six to seven iron atoms that are donated to Isu1p, in the early stages of Fe/S cluster assembly (18). A physical interaction between Yfh1p and Isu1p in yeast has been demonstrated (25,26). Therefore, it has been proposed that Yfh1p has ferroxidase activity and iron storage properties which may protect the mitochondria from iron toxicity, and that it also acts as a chaperone to donate iron to the proteins involved in the two major pathways of iron utilization, Fe/S cluster assembly and heme synthesis. The relative importance of these two functions cannot be easily inferred by the effects of Yfh1p deficiency. In fact, yeast cells lacking frataxin (yfh1{Delta}) show a rather complex respiratory-deficient phenotype that results from a massive mitochondrial iron deposition, defects in the maturation of mitochondrial Fe/S enzymes, hypersensitivity to oxidative stress, instability of mtDNA (27–29) and defects in heme synthesis (24). Mitochondrial iron overload was prevented by growth in low iron media (30), deletion of genes for high-affinity iron transport (31) or increased vacuolar iron transport (32). Under these conditions, respiratory activity was preserved, although the activity of Fe/S enzymes remained sub-optimal (33). Similarly, zinc prevented iron accumulation in the mitochondria of these strains, but not the deficiency of Fe/S and heme proteins (34). It has been proposed that the sub-optimal biosynthesis of Fe/S clusters is the direct cause of mitochondrial iron overload and of high sensitivity to oxidative damage (28). However, the primary target of Yfh1p deficiency might be proper mitochondrial iron handling.

We thought that a direct comparison of MtF and frataxin/Yfh1p may be useful to identify differences in their biological activity, considering that both are mitochondrial iron-binding proteins, but MtF is not expected to be able to interact specifically with Isu1p and ferrochelatase, as observed for Yfh1p. To this aim we expressed MtF in yeast cells lacking Yfh1p. The results show that MtF rescues most of the defects associated with Yfh1p deficiency, and that it prevents mitochondrial iron overload. The data suggest that the primary role of frataxin is to sequester mitochondrial iron in order to prevent its toxicity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of MtF in wild-type yeast
The cDNA for the full-length precursor of human MtF was subcloned into a high-copy expression plasmid, pG3/TRP1 (35), downstream of the constitutively active yeast glyceraldehyde-3-phosphate dehydrogenase promoter. The resulting plasmid (pG3-MtF/TRP1) was used to transform the haploid yfh1{Delta} [YCp50–YFH1/URA3] strain, which contains a disrupted yfh1{Delta}::HIS3 allele and a complementing YCp50–YFH1 plasmid for the constitutive expression of Yfh1p, the yeast homolog of frataxin (36). The new strain was designated [YFH1+huMtF], whereas a strain carrying empty pG3/TRP1 plasmid, yfh1{Delta} [YCp50–YFH1/URA3][pG3-/TRP1], was designated [YFH1] and considered a control strain (Table 1). Immunoblotting with anti human MtF revealed a band of the expected mobility in the homogenates of the transformed cells, which was absent in the non-transformed parent strain (Fig. 1A, lanes 3 versus 2). The electrophoretic mobility of the assembled human MtF expressed in yeast and HeLa cells was the same, and the levels of expression in the two cell types were comparable (Fig. 1A, lanes 1 and 3). The transformed yeast cells were incubated for 18 h with 5 µM [55Fe] ferric ammonium citrate (FAC), and the homogenates separated on non-denaturing PAGE and exposed to autoradiography. A single band co-migrating with MtF was evident (Fig. 1A, lane 5), showing that the transfectant ferritin is functional in taking up iron within yeast cells. To verify its cellular localization, we separated the mitochondrial from the post-mitochondrial fractions by differential centrifugation, using succinate dehydrogenase (SDH) activity as mitochondrial marker (data not shown). Immunoblotting showed that MtF accumulated specifically in the mitochondria; a minor signal found in the cytoplasm was attributed to contamination of this fraction (Fig. 1B). We conclude that the transformed yeast cells efficiently express human MtF precursor, which is correctly processed in the mitochondria to mature and functional ferritin molecules, as it occurs in mammalian cells.


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  		Table 1. S. cerevisiae strains used in this work
 


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  		Figure 1. Human MtF expression in yeast. (A) Yeast strains were grown in SSDE media alone or supplemented with 5 µM 55Fe. The cells were lysed using glass beads in the presence of 1% Triton X-100, and 30 µg of the total soluble protein preparation were heated at 70°C for 10 min and centrifuged for 5 min at 10 000g. The resulting soluble fractions were loaded on non-denaturing PAGE, blotted and assayed by anti-MtF antibody (lanes 1–4) or exposed to autoradiography (lanes 5 and 6). Lane 1: HeLa MtF over-expressing clone, lane 2: untransformed yeast, lanes 3 and 5: yeast transformed with both [YCp50–YFH1] and [pG3-MtF/TRP1] plasmids, lanes 4 and 6: [huMtF] strain. (B) Yeast strains were grown in SSDE media and the spheroplasts obtained by zymolyase treatment. Mitochondrial fraction was extracted by digitonin followed by sequential centrifugation. 5 µg of mitochondrial (MF) and 20 µg of post-mitochondrial fractions (PMF) of yeast transformed with pG3-MtF/TRP1 were assayed with anti-MtF antibody as in (A). The arrows indicate human MtF.

 
Expression of MtF in yfh1{Delta} yeast strains
The expression of human MtF did not confer any evident phenotype to the yfh1{Delta} [YCp50–YFH1/URA3] strain under standard growth conditions. We considered that MtF functionality might become more evident in a non-complemented yfh1{Delta} background in which mitochondrial iron homeostasis is altered due to the lack of Yfh1p. Therefore, the YCp50–YFH1/URA3 plasmid was eliminated by treatment with 5-fluoroorotic acid (5-FOA). Several clones were subjected to various rounds of counter-selection with 5-FOA yielding yfh1{Delta} cells that only contained the pG3-MtF/TRP1 plasmid. Three isolates, originating from independent transformants, were selected for further characterization and named [huMtF]. Analogous treatment was used to obtain yfh1{Delta} cells containing empty pG3-/TRP1 plasmid ({Delta} [YFH1] strain). Some of the properties of the three strains used in this work are summarized in Table 1. The complete elimination of the YCp50–YFH1 plasmid was verified by PCR amplification of total yeast DNA using YFH1-specific primers (Fig. 2A), whereas MtF expression was tested by immunoblotting (Fig. 2B). The levels of MtF protein and 55Fe incorporated into MtF were about 2-fold higher in the presence than in the absence of Yfh1p (Fig. 1A, lanes 3–6). This finding indicates that the lack of Yfh1p does not affect MtF iron incorporation activity, although it modifies MtF protein levels.



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  		Figure 2. Analysis of Yfh1p and MtF expression. (A) The presence of YFH1 gene was analyzed by PCR amplification using the total DNA preparations from yeast [YFH1], {Delta} [YFH1] and [huMtF] clones. YFH1 gene produced a 240 bp fragment whereas the RPO22 nuclear gene used as a control produced a 637 bp fragment. Oligonucleotides and procedures are specified in Materials and Methods. (B) Western blotting of total protein preparations treated as in (A) of Figure 1. Three different [huMtF] clones were assayed for MtF expression in comparison with untransformed [YFH1] strain. The arrow indicates the correctly assembled MtF.

 
MtF expression rescues the growth defect caused by the loss of Yfh1p
Yfh1p-deficient yeast cells typically show slow or absent growth on non-fermentable carbon sources. We therefore analysed whether MtF expression might modify this property. Cells grown on selective synthetic dextrose essential (SSDE) plates were diluted in SSDE liquid medium to 0.03 OD600 nm and the growth was followed for 72 h. The {Delta} [YFH1] strain took about 70 h to reach the stationary phase, whereas the three [huMtF] clones and the [YFH1] strain took only ~20 h (Fig. 3A). In other experiments, the growth of the different strains in SSDE liquid medium was synchronized at the exponential phase. Then they were diluted to 0.2 OD600 nm and each strain was spotted on plates and grown for 4 days at 30°C. All strains grew to a similar extent on complete solid medium containing a fermentable carbon source (dextrose, YPD), as expected. In contrast, the growth of {Delta} [YFH1] strain was severely reduced on non-fermentable carbon sources (ethanol and glycerol, YPEG), whereas that of the three [huMtF] clones was comparable to that of the control [YFH1] strain (Fig. 3B). Next we verified whether the rescue of respiratory activity in the [huMtF] strain could be attributed to MtF expression. To this aim, the [huMtF] 1,2,3 clones were maintained in non-selective completed liquid medium for several generations. We obtained the corresponding {Delta} [huMtF] A,B,C clones lacking the pG3-MtF plasmid, which were unable to grow on plates lacking tryptophan. The absence of the pG3-MtF/TRP1 plasmid in these clones was verified by PCR analysis using primers specific for MtF cDNA (Fig. 4A). As expected, {Delta} [huMtF] A,B,C clones grew on YPD plates but were unable to proliferate on YPEG plates (Fig. 4B). We conclude that the respiratory activity of the [huMtF] clones is associated with the expression of MtF.




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  		Figure 3. [huMtF] clones grow on non-fermentable carbon sources. (A) The rate of cellular growth was evaluated by optical density (OD). Cells were harvested from SSDE plates, diluted to a concentration of 0.03 OD600 nm in SSDE liquid media and the growth was followed for 72 h. [YFH1], diamonds; {Delta} [YFH1], squares and [huMtF], triangles. The clones are the ones described in Figure 2. The experiments were repeated at least four times, always with different clones from each strain, and the three [huMtF] clones were used, always obtaining similar results. A representative experiment is shown. (B) The growth of the indicated clones in SSDE liquid media was synchronized at the exponential growth phase. Then cells were diluted to 0.2 OD600 nm and equally spotted on YPD and YPEG plates or SD plates supplemented with essential nutrients with or without uracil (SSDE). Pictures were taken after 4 days of growth at 30°C. This analysis was repeated three times with similar results, and a representative one is shown.

 


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  		Figure 4. Deletion of MtF plasmid in [huMtF] inhibits the growth on non-fermentable carbon sources. Three different clones of [huMtF] strain were selected for the inability to grow on selective SD plates without L-tryptophan. (A) The loss of the MtF plasmid in these {Delta} [huMtF] clones was verified by PCR amplification using specific primers. A 281 bp fragment inside the sequence of MtF gene was amplified in parallel with a 637 bp fragment of RPO22 nuclear gene as control. (B) The growth of the indicated clones was synchronized in their SSDE liquid media, and the cells spotted on different solid media as shown in Figure 3.

 
MtF expression and mitochondrial functionality
The activity of mitochondrial iron–sulfur enzymes is abnormally low in yf h1{Delta} strains. We analyzed whether MtF expression might correct this defect. Cells were grown in minimal SSDE medium with or without supplementation with 15 µM iron provided as FAC. The cells were harvested after they reached the exponential growth phase and mitochondria were isolated and analyzed. The activity of malate dehydrogenase (MDH) was used as a control and was detected at similar levels in all the strains analyzed and under both experimental conditions (Fig. 5). As expected, the specific activities of the Fe/S enzymes, aconitase (ACO) and SDH, were strongly reduced in the {Delta} [YFH1] strain, and almost fully inhibited in medium supplemented with FAC (Fig. 5). Interestingly, ACO activity was present at similar levels in the [huMtF] and the control [YFH1] strains (Fig. 5). Immunoblotting with anti-yeast ACO antibodies showed that the {Delta} [YFH1], [YFH1] and [huMtF] strains contained similar ACO protein levels both in the absence and in the presence of FAC (data not shown). SDH activity was ~50% lower in the [huMtF] strain when compared with the [YFH1] strain under either growth condition (Fig. 5). In addition, SDH activity was reduced to ~80% of control levels in the [YFH1+huMtF] strain (Fig. 5). Together, these findings indicate that MtF expression protects the activity of mitochondrial Fe/S enzymes, probably against iron-induced oxidative damage.



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  		Figure 5. The activity of Fe/S enzymes is partially maintained in [huMtF] clones. The growth of the indicated clones was synchronized at exponential growth phase in SSDE liquid media, with or without addition of 15 µM FAC. Mitochondrial extracts were prepared and the activity of aconitase and SDH was measured. The activity of MDH was measured as a control of mitochondrial non-iron–sulfur cluster enzyme. The activities, measured as nmol of substrate consumed by milligram of mitochondrial protein per minute, were normalized on the control [YFH1] clone. The data are the means and SD of five independent measures for aconitase and MDH activity and of seven for SDH. The experiments were repeated twice on cells grown in basic medium and three times grown in medium supplemented with FAC and the figure shows one representative experiment for every growth condition.

 
To study possible MtF effects on mitochondrial iron levels, the cells were grown in SSDE media supplemented with 15 µM FAC until they reached 1 OD600nm/ml. Then they were harvested, and the mitochondrial and post-mitochondrial fractions were separated and analyzed for iron content by a colorimetric method. Mitochondrial iron levels were increased 6–8-fold in {Delta} [YFH1] relative to [YFH1], as expected, but only 2-fold in [huMtF] (Table 2). To analyze iron compartmentalization in the mitochondria of the [huMtF] strain, the cells were grown in SSDE media supplemented with 5 µM 55Fe, and the radioactivity in the mitochondrial fraction counted before and after immunoprecipitation with anti-MtF antibodies. In two experiments we found that 50–55% of mitochondrial iron was associated with MtF.


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  		Table 2. Mitochondrial iron content
 
It was reported that a secondary effect of mitochondrial iron overload is instability of mtDNA, which is typically damaged in yfh1{Delta} strain (29). To study this, the cells were maintained in log phase of growth for 48 h in YPD liquid medium. Then, DNA integrity was verified by PCR amplification of fragments of the mitochondrial gene OLI1 and of the nuclear gene RPO22, used as an internal control. Whereas the RPO22 gene was equally amplified in the different strains, the mitochondrial gene OL11 could only be amplified in the control [YFH1] and in the [huMtF] strains, but not in the {Delta} [YFH1] and {Delta} [huMtF] strains (Fig. 7).



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  		Figure 7. Expression of MtF preserves mitochondrial DNA. Clones of the indicated strains were maintained in log phase growth for 48 h in YPD complete liquid medium. The presence of mtDNA in the total DNA preparations was analyzed by PCR amplification of the OLI1 gene, which produced a 189 bp fragment. PCR amplification of RPO22 nuclear gene, which produced a 637 bp fragment, was run in parallel as a control.

 
MtF expression and resistance to H2O2
Yeast cells lacking frataxin were previously found to have a reduced resistance to H2O2 treatment compared with controls (37). To assess whether huMtF might increase anti-oxidant resistance, the [huMtF], [YFH1] and {Delta} [YFH1] strains were grown in SSDE media and synchronized at the exponential phase of growth. Then, equal numbers of cells were spotted on YPD plates and exposed to H2O2 gradients. The cells were grown for 4 days at 30°C, and analyzed. The higher sensitivity of the {Delta} [YFH1] strain was particularly evident when a larger amount of H2O2 was loaded in the middle of the plates (Fig. 6, right side). In contrast, the [huMtF] strain was as affected by H2O2 as the frataxin-expressing [YFH1] strain. This result confirms that MtF expression has a protective role under conditions of increased oxidative damage.



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  		Figure 6. [huMtF] clones are resistant to H2O2. The growth of the indicated strains was synchronized at the exponential growth phase in SSDE media; the cells were diluted as in Figure 4 and spotted on YPD plates. Gradients of H2O2 were generated by adding to a filter in the middle of the plate 20 and 40 µl of 30% H2O2. The picture was taken after 4 days of growth at 30°C. The experiment was repeated five times, with different [huMtF] clones, producing the same results, and a representative one is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The major function of cytosolic ferritins is to maintain a supply of bioavailable and non-toxic iron in the cytosol and to protect cells from iron excess (1). To verify whether MtF has the same function inside the mitochondrion we used a well-characterized model of mitochondrial iron overload: a yeast strain deficient in frataxin/Yfh1p. Yeasts are among few organisms that do not express ferritins but use alternative methods for iron storage (38). However, H- and L-ferritins have already been expressed in yeast cells, where they accumulate as functional iron-incorporating proteins (39,40). Therefore, the finding that MtF was efficiently expressed in S. cerevisiae was expected. We found specific MtF accumulation in the mitochondria, indicating that mitochondrial import and processing of the MtF precursor occurred correctly as in mammalian cells. This was also not surprising given that other mammalian mitochondrial proteins, including human frataxin (37), were previously expressed in yeast and efficiently targeted to the mitochondria. MtF incorporated iron in yeast, confirming that the protein is functional and has access to the metal as in mammalian mitochondria. We conclude that yeast is a suitable model to study MtF functionality.

MtF partially rescues the defects caused by frataxin deficiency
Yeast cells lacking Yfh1p are characterized by a slow-growth phenotype (petite), an inability to grow on non-fermentable carbon sources such as ethanol or glycerol, and a high sensitivity to oxidative stress caused by H2O2 (37). When we expressed MtF in these cells (i.e. the [huMtF] strain), we first observed that their growth rate in glucose-containing media was similar to that of the control [YFH1] strain (Fig. 3). Even more evident was the effect when cells were grown in the presence of non-fermentable carbon sources. The {Delta} [YFH1] strain did not grow at all, whereas [huMtF] grew similar to the control cells (Fig. 3). That this effect was due to MtF expression was confirmed by showing that loss of the plasmid encoding MtF resulted in inability to grow on YPEG (Fig. 4). We conclude that MtF restored the ability of yfh1{Delta} to grow under conditions in which mitochondrial respiratory activity is required.

Next, we analyzed the activity of the Fe/S enzymes, ACO and SDH, under different conditions. When {Delta} [YFH1] cells were grown in media supplemented with 15 µM iron (the concentration found in the YPD medium) the activity of both enzymes was almost fully suppressed (Fig. 5), whereas in MtF-expressing cells ACO activity was essentially normal and SDH activity was equal to about 50% of controls (Fig. 5). These findings indicate that MtF expression corrects the decrease in Fe/S enzyme activity associated with Yfh1p-deficiency. It is known that cellular iron deprivation can also protect the activity of Fe/S enzymes in Yfh1p deficient cells; for example, ACO and SDH activities were detectable at about 20–30% of controls in cells grown in iron-limiting media (Fig. 5). Similarly, in the Yfh1p-deficient cells in which iron availability was limited by chelators (30) or genetic alterations (31,32). ACO activity was about 60% of controls, whereas we obtained full ACO reactivation. These data suggest that the mechanism by which MtF rescues Fe/S enzyme activities differs from cellular iron deprivation.

Oxidative stress is a hallmark of Yfh1p deficiency when the cells are grown in iron-containing media (30). This can be assessed by the integrity of mtDNA, which is one of the targets of ROS formation (29). We have shown that under conditions in which mtDNA was degraded and undetectable in {Delta} [YFH1] cells, it remained detectable in the [huMtF] strain. Loss of the MtF plasmid abolished this protection, confirming that it was associated with MtF expression. That MtF protects the mitochondrion from oxidative damage is further supported by the [huMtF] strain resistance to H2O2, which was much higher than that of the {Delta} [YFH1] strain, and similar to that of the control [YFH1] strain. This is consistent with the hypothesis that MtF sequesters local iron excess and prevents Fenton-like reactions.

As expected, Yfh1p deficiency was associated with a high ~8-fold increase in mitochondrial iron content, whereas, surprisingly, we found only ~2-fold increase in the MtF-expressing strain (Table 2). This is an apparent paradox since MtF is an iron storage protein with a large capacity for metal binding, and was expected to sequester the excess iron normally taken up by the Yfh1p-deficient mitochondria. In fact, the observed 2-fold increase was likely caused by the iron sequestering activity of MtF, since we found that MtF-iron corresponded to about 50% of the total mitochondrial iron in the [huMtF] strain. Similar findings were obtained in mammalian cells, where MtF expression increased the level of mitochondrial iron (9). Given that inhibition of Fe–S cluster synthesis decreases mitochondrial iron export (33), it is possible that the absence of mitochondrial iron deposition in [huMtF] cells results from MtF-mediated protection of Fe–S clusters and/or enzymes involved in Fe–S cluster synthesis.

Function of MtF and Yfh1p
We found that MtF can efficiently replace the function of Yfh1p in yeast, but these two proteins have different structures and characteristics, although they both bind iron. Ferritin iron-binding properties have been largely characterized and the reaction of iron incorporation has been elucidated (1,41). It involves transient iron-binding at the catalytic site of the H-subunit, followed by oxidation, movement inside the large cavity and mineralization. The mineral iron core is inactive in Fenton reaction, and becomes bioavailable only after reduction or degradation of the protein shell. Yfh1p iron-binding properties seem to be more complex. The protein also binds iron transiently in a manner that is made available to Isu1p, ferrochelatase or [3Fe–4S] aconitase, thus acting as a chaperone (18,21). In addition, if Fe(II) is not transferred from Yfh1p to other ligands, it is oxidized and stored inside the protein oligomers, thus acting as an iron storage molecule (22,23). A common property of the two molecules seems to be the capacity to bind iron transiently, a property that is intrinsically difficult to detect experimentally. Easier to follow is the iron incorporating activity of ferritin, and that of MtF did not seem to be affected by the presence or absence of Yfh1p (Fig. 1A). This indicates that Yfh1p function was not replaced by the capacity of MtF to incorporate iron in the cavity. In addition, it seems unlikely that ferritin can substitute Yfh1p chaperone functions, although it has been observed that the iron oxidized by H-ferritin can be donated to molecules with high affinity for Fe(III) such as transferrin (1). We postulate that the more likely MtF function in this context is the capacity to bind iron transiently and possibly to oxidize it. This would control the size of labile iron that is potentially toxic.

Our data can be interpreted as follows: in the absence of the metal binding sites offered by Yfh1p, an excess of labile mitochondrial iron accumulates, which is toxic and induces oxidative stress. This affects not only mtDNA and Fe/S enzymes (Figs 5 and 7), but also the proteins involved in the synthesis of Fe/S clusters. In fact some of them (e.g. IscU, Yah1p) are Fe/S proteins and likely to be easy targets of oxidative damage. The inactivation of any of these proteins has been found to cause iron overload (42), with a mechanism which might be similar to the one occurring in genetic sideroblastic anemia, where the inefficient utilization of iron caused by ALAS2 deficiency increases mitochondria iron influx (12). The expressed MtF may bind and oxidize the toxic iron, thus inhibiting the initiation of oxidative damage and the vicious cycle that results in the inactivation of Fe/S synthesis and damage to respiratory enzymes. This mechanism would also explain why MtF expression inhibits iron deposition in the mitochondria. Moreover, MtF iron-binding/oxidizing activity seems to be much higher than that of Yfh1p. For example, the rate of in vitro iron oxidation was found to be several fold faster in the presence of H-ferritin than in the presence of Yfh1p (22), and the affinity for Fe(II) is expected to be higher for ferritin than Yfh1p, although no direct comparison has been made so far. In addition, MtF expression, directed by a high-copy number plasmid, is predicted to be higher than that of Yfh1p. This would suggest that the level of labile iron in the presence of MtF is lower than that in the presence of Yfh1p. This observation might partially explain why the activity of SDH in the strains expressing MtF was lower than that of the control. SDH has a higher number of iron atoms than ACO, and therefore might be more sensitive to the relative iron deficiency caused by MtF expression.

Implications for mammalian cells
The major observation of this work is that an iron-binding protein such as MtF can substitute in large extent the function of frataxin in yeast, and that mitochondrial iron overload is probably secondary to the formation of minor amounts of toxic iron. We propose that the major role of Yfh1p, which can be substituted for by MtF, is transient iron-binding and detoxification. These observations are in partial agreement with data in mammalian cells, where heavy mitochondrial iron overload has been rarely observed, and in the cardiomyocytes of a mouse model for FRDA, where tissue damage occurred before evident iron loading (43). In the FRDA mouse models that develop ataxia, iron deposition was not observed in the damaged tissues, confirming that this is not necessarily associated with frataxin deficiency (44). However, a reduction of the activity of SDH and Fe/S enzymes, and an increase in oxidative stress are consistent findings in tissues and cultured fibroblasts from FRDA patients (45). This might be caused by the local deregulation of mitochondria iron, as we propose to occur in yeast, caused by catalytic concentrations of iron that cannot be detected. Alternatively, it is possible that the iron chaperone function of frataxin is more important in mammals than in yeast. The study of MtF expression in frataxin-deficient mammalian cells may help to clarify this point. Our preliminary data indicate that MtF has a general protective role against oxidative damage in cultured mammalian cells (data not shown). So far we do not have any direct evidence that MtF is upregulated in frataxin-deficient cells, but we may expect that its artificial upregulation may be protective against the damage.

In conclusion the present results indicate that the MtF expression strongly protects yeast cells from damage associated with frataxin deficiency. These data could be relevant not only for FRDA but also for other neurodegenerative disease, where an increase in the ROS production within the mitochondria has been showed to be implicated in the pathogenesis of neurodegeneration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid construction
The cDNA of human MtF precursor was PCR amplified from the vector pUHD-MtF (9) using the primers 5'-GCCGCCGGTACCGTAGCAATGCTGTCCTGC with a Sal I site and a yeast ribosomal binding site, and 5'-GCCGCCGTCGACTTAGTTCTGCTTGTTTTC with a Kpn I site. The cDNA was subcloned into pG3/TRP1 vector (35) downstream the yeast glyceraldehyde-3-phosphate dehydrogenase promoter to obtain the construct pG3-MtF/TRP1 for constitutive expression in S. cerevisiae.

Yeast strains and culture
All strains used in this study were derived from strain yfh1{Delta} [YFH1], a haploid derivative of strain YPH501, carrying a disrupted chromosomal yfh1{Delta}::HIS3 allele and a complementing Ycp50–YFH1 plasmid (36). It was maintained in SD minimal medium supplemented with adenine (40 µg/ml), L-leucine (60 µg/ml), L-lysine (30 µg/ml) (Selective SD medium, SSD) and L-tryptophan (40 µg/ml). This strain was transformed with the 2 µm vectors pG3/TRP1 or pG3-MtF/TRP1, and four transformants for each vector were subjected to counter-selection with 5-FOA, to eliminate the URA3-based Ycp50–YFH1 plasmid, as described (36). Over 10 independent clones that grew on 5-FOA-containing plates were picked and analyzed for growth on YPD and YPEG, and representative clones, designated {Delta} [YFH1] and [huMtF]1,2,3, were maintained in SSD solid medium supplemented with uracil, and used in all experiments. Strain yfh1{Delta} [YCp50–YFH1/URA3][pG3-/TRP1] was named [YFH1] and maintained in SSD. To abolish MtF expression in the [huMtF] strain (yfh1{Delta} [pG3-MtF/TRP1]) cells were grown for several generations in non-selective complete liquid medium to allow for random loss of the pG3-MtF/TRP1 plasmid. Representative clones unable to grow on plates lacking tryptophan were designated {Delta} [huMtF] and maintained on SSD plates supplemented with uracil and tryptophan. In some experiments, the cells were grown in SSD liquid media, containing the appropriate supplements (SSDE), with or without addition of 15 µM FAC, which is the iron concentration measured in YPD medium. YDP, containing 2% dextrose, and YPEG, containing 3% glycerol and 2% ethanol, were used to test growth on fermentable and non-fermentable carbon sources, respectively. To analyze resistance to H2O2, circular filter papers soaked with 20 or 40 µl of 30% H2O2 were placed in the middle of YPD plates to generate gradient concentrations.

Cell extracts preparations
Cells were harvested by 5 min centrifugation at 1500g and washed with ice-cold water. For DNA extraction, the cells were suspended in 10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1% Triton X-100, phenol–chloroform (1 : 1). After vortexing at maximum speed for 2 min in the presence of glass beads, the samples were centrifuged and the aqueous phase was collected and used for DNA amplification. To prepare total soluble homogenates, the cells were suspended in 20 mM Tris–HCl, pH 7.5, 1% Triton X-100, and vortexed at maximum speed for 5 min in the presence of glass beads. The supernatants upon centrifugation at 10 000g for 10 min were collected. Spheroplasts were obtained by suspending the cells in 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 M sorbitol, adding 200 U of zymolyase per ml of original packed cell volume, incubating for 1 h at 30°C with gentle agitation and collected by centrifugation at 1000g for 5 min. Mitochondria were isolated by suspending the spheroplasts in 10 mM HEPES–NaOH, pH 7.2, 0.6 M mannitol, 1 mM PMSF and 0.007% digitonin (mitochondrial buffer) and incubated in gentle agitation for 20 min at 4°C. The preparations were then centrifuged at 600g for 3 min and the supernatants collected. The precipitates were resuspended in mitochondrial buffer and incubated in gentle agitation for 5 min at 4°C and centrifuged. The two supernatants were pooled and then centrifuged at 7000g for 10 min. The precipitates were gently suspended in mitochondrial buffer, centrifuged at 600g for 3 min, to remove membrane aggregates, and the supernatants centrifuged again at 7000g for 10 min to collect mitochondria-enriched pellets. Finally the preparations were suspended and washed two times in mitochondrial buffer.

Enzymatic activities
Fresh mitochondria preparations were suspended in 10 mM HEPES–NaOH, pH 7.2, 20 mM KCl, 5 mM KPO4, 220 mM sucrose and aconitase, SDH and MDH activities were measured following standard procedures (46–48). Aconitase was assayed by measuring the disappearance of cis-aconitate at 240 nm, SDH by following the reduction of para-iodonitrotetrazolium violet (INT) to INT–formazan at 500 nm, and MDH by monitoring disappearance of NADH at 340 nm.

Protein analysis
Protein concentration was measured using BCA assay (Pierce) calibrated on bovine serum albumin. The mitochondrial preparations were diluted in 20 mM Tris–HCl, pH 7.5, 1% Triton X-100 before BCA assay. Before western blotting, the extracts were heated 10 min at 75°C to extract the ferritin, which is thermo resistant, and centrifuged at 10 000g for 5 min. The supernatants were separated on non-denaturing 6% polyacrylamide gel electrophoresis in Tris–glycine buffer. Proteins were transferred to Hybond ECL nitrocellulose membranes (Amersham) with a semidry blotting apparatus. The membranes were incubated with mouse antiserum anti-huMtF diluted 1 : 1000 and with secondary, peroxidase labeled, anti-mouse IgG diluted 1 : 5000 (Sigma) or with anti-yeast Aco1p (a generous gift from Professor Roland Lill) diluted 1 : 3000, followed by a peroxidase labeled anti-rabbit IgG 1 : 5000 (Sigma). Bound activity was revealed with Super Signal West Pico system (Pierce).

55Fe labeling and total mitochondrial iron determination
Yeast cells were grown in SSDE medium added of 5 µM 55Fe-citrate and 100 µM ascorbic acid for 18 h. The soluble homogenates were heated, centrifuged and separated as for western blotting analysis and the gels were exposed to autoradiography. Iron content was determined as described earlier (49). Briefly, mitochondrial and cytoplasm preparations were incubated in 100 mM thioglycolic acid, 20 mM HCl and 20 mM 2,2'-bipyridyl, the samples were boiled for 20 min and analyzed for absorbance at 570 nm, subtracting the absorbance at 465 nm, to quantify the Fe–2,2'-bipyridyl complex. Mitochondrial preparation from [huMtF] strain was immunoprecipitated as described earlier (9). Samples of 10 µl before and after immunoprecipitation were added of 500 µl of liquid scintillation cocktail (Packard), incubated in dark for 20 min and finally counted.

DNA analysis
Nuclear, mitochondrial and plasmidic DNAs of yeast strains were PCR amplified using standard procedures, with the following oligonucleotides primers: YFH1 gene, 5'-CTCATCCGGATTGTATACCT-3' and 5'-GGCTTTTAGAAATGGCCTTC-3'; OLI1 gene, 5'-TAAATATATTGGAGCAGGTATC-3' and 5'-GAATGAAACCATTAAACAGAATA-3' RPO22, 5'-AAACACCTGCCCTAGAAGAC-3' and 5'-GTCGTGAGGGTAGTTCAGC-3' and HuMtF: 5'-CCGGGAGAAAACCGAGGGCGCGGAGAA-3' and 5'-GCACGTGGTCACCTAGTTC-3'.


   ACKNOWLEDGEMENTS
 
We are grateful to Professor Roland Lill for the kind gift of anti-yeast Aco1p antibody. This work was partially supported by grants of the Fondazione Mariani to P.A. and of Telethon- Italia GP0075Y01 to S.L., and in part by grants AG15709 to G.I. and NRSA 44748 to H.A.O. from the National Institutes of Health.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +39 0226464755; Fax: +39 0226434844; Email: levi.sonia{at}hsr.it


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Harrison, P.M. and Arosio, P. (1996) The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta, 1275, 161–203.[Medline]

  2. Hentze, M.W. and Kuhn, L.C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress. Proc. Natl Acad. Sci. USA, 93, 8175–8182.[Abstract/Free Full Text]

  3. Arosio, P. and Levi, S. (2002) Ferritin, iron homeostasis and oxidative damage(1,2). Free Radic. Biol. Med., 33, 457–463.[CrossRef][Web of Science][Medline]

  4. Ponka, P. (1997) Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells. Blood, 89, 1–25.[Abstract/Free Full Text]

  5. Lill, R. and Kispal, G. (2000) Maturation of cellular Fe–S proteins: an essential function of mitochondria. Trends Biochem. Sci., 25, 352–356.[CrossRef][Web of Science][Medline]

  6. Levi, S., Corsi, B., Bosisio, M., Invernizzi, R., Volz, A., Sanford, D., Arosio, P. and Drysdale, J. (2001) A human mitochondrial ferritin encoded by an intronless gene. J. Biol. Chem., 270, 24437–24440.

  7. Drysdale, J., Arosio, P., Invernizzi, R., Cazzola, M., Volz, A., Corsi, B., Biasiotto, G. and Levi, S. (2002) Mitochondrial ferritin—a new player in iron metabolism. Blood Cells Mol. Dis., 229, 376–383.

  8. Langlois d'Estaintot, B., Santambrogio, P., Granier, T., Gallois, B., Chevalier, J.M., Précigoux, G., Levi, S. and Arosio, P. (2004) Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala. J. Mol. Biol., 340, 277–293.[CrossRef][Web of Science][Medline]

  9. Corsi, B., Cozzi, A., Arosio, P., Drysdale, J., Santambrogio, P., Campanella, A., Biasiotto, G., Albertini, A. and Levi, S. (2002) Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects cellular iron metabolism. J. Biol. Chem., 277, 22430–22437.[Abstract/Free Full Text]

  10. Levi, S. and Arosio, P. (2004) Mitochondrial ferritin: an iron storage protein inside the mitochondria. Int. J. Biochem. Cell Biol. 36, 1887–1889.[CrossRef][Web of Science][Medline]

  11. Cazzola, M., Invernizzi, R., Bergamaschi, G., Levi, S., Corsi, B., Travaglino, E., Rolandi, V., Biasiotto, G., Drysdale, J. and Arosio, P. (2003) Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia. Blood, 101, 1996–2000.[Abstract/Free Full Text]

  12. Alcindor, T. and Bridges, K.R. (2002) Sideroblastic anaemias. Br. J. Haematol., 116, 733–743.[CrossRef][Web of Science][Medline]

  13. Gattermann, N. (2000) From sideroblastic anemia to the role of mitochondrial DNA mutations in myelodysplastic syndromes. Leuk. Res., 24, 141–151.[CrossRef][Web of Science][Medline]

  14. Patel, P.I. and Isaya, G. (2001) Friedreich ataxia: from GAA triplet-repeat expansion to frataxin deficiency. Am. J. Hum. Genet., 69, 15–24.[CrossRef][Web of Science][Medline]

  15. Pandolfo, M. (2003) Friedreich ataxia. Semin. Pediatr. Neurol., 10, 163–172.[CrossRef][Medline]

  16. Dhe-Paganon, S., Shigeta, R., Chi, Y.I., Ristow, M. and Shoelson, S.E. (2000) Crystal structure of human frataxin. J. Biol. Chem., 275, 30753–30756.[Abstract/Free Full Text]

  17. Musco, G., Stier, G., Kolmerer, B., Adinolfi, S., Martin, S., Frenkiel, T., Gibson, T. and Pastore, A. (2000) Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin. Structure Fold Des., 8, 695–707.[Medline]

  18. Yoon, T. and Cowan, J.A. (2003) Iron–sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe–2S] clusters in ISU-type proteins. J. Am. Chem. Soc., 125, 6078–6084.[CrossRef][Web of Science][Medline]

  19. Adamec, J., Rusnak, F., Owen, W.G., Naylor, S., Benson, L.M., Gacy, A.M. and Isaya, G. (2000) Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia. Am. J. Hum.Genet., 67, 549–562.[CrossRef][Web of Science][Medline]

  20. Cavadini, P., O'Neill, H.A., Benada, O. and Isaya, G. (2002) Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia. Hum. Mol. Genet., 11, 217–227.[Abstract/Free Full Text]

  21. Park, S., Gakh, O., O'Neill, H.A., Mangravita, A., Nichel, H., Ferriera, G.C. and Isaya, G. (2003) Yeast frataxin sequentially chaperones and stores iron by coupling protein assembly with iron oxidation. J. Biol. Chem., 278, 31340–31351.[Abstract/Free Full Text]

  22. Park, S., Gakh, O., Mooney, S.M. and Isaya, G. (2002) The ferroxidase activity of yeast frataxin. J. Biol. Chem., 277, 38589–38595.[Abstract/Free Full Text]

  23. Nichol, H., Gakh, O., O'Neill, H.A., Pickering, I.J., Isaya, G. and George, G.N. (2003) Structure of frataxin iron cores: an X-ray absorption spectroscopic study. Biochemistry, 42, 5971–5976.[CrossRef][Medline]

  24. Lesuisse, E., Santos, R., Matzanke, B.F., Knight, S.A., Camadro, J.M. and Dancis, A. (2003) Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1). Hum. Mol. Genet., 12, 879–889.[Abstract/Free Full Text]

  25. Ramazzotti, A., Vanmansart, V. and Foury, F. (2004) Mitochondrial functional interactions between frataxin and Isu1p, the iron–sulfur cluster scaffold protein, in Saccharomyces cerevisiae. FEBS Lett., 557, 215–220.[CrossRef][Web of Science][Medline]

  26. Gerber, J., Muhlenhoff, U. and Lill, R. (2003) An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1. EMBO Rep., 4, 906–911.[CrossRef][Web of Science][Medline]

  27. Babcock, M., de Silva, D., Oaks, R., Davis-Kaplan, S., Jiralerspong, S., Montermini, L., Pandolfo, M. and Kaplan, J. (1997) Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin. Science, 276, 1709–1712.[Abstract/Free Full Text]

  28. Muhlenhoff, U., Richhardt, N., Ristow, M., Kispal, G. and Lill, R. (2002) The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins. Hum. Mol. Genet., 11, 2025–2036.[Abstract/Free Full Text]

  29. Karthikeyan, G., Santos, J.H., Graziewicz, M.A., Copeland, W.C., Isaya, G., Van Houten, B. and Resnick, M.A. (2003) Reduction in frataxin causes progressive accumulation of mitochondrial damage. Hum. Mol. Genet., 12, 3331–3342.[Abstract/Free Full Text]

  30. Foury, F. (1999) Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain. FEBS Lett., 456, 281–284.[CrossRef][Web of Science][Medline]

  31. Radisky, D.C., Babcock, M.C. and Kaplan, J. (1999) The yeast frataxin homologue mediates mitochondrial iron efflux. Evidence for a mitochondrial iron cycle. J. Biol. Chem., 274, 4497–4499.[Abstract/Free Full Text]

  32. Chen, O.S. and Kaplan, J. (2000) CCC1 suppresses mitochondrial damage in the yeast model of Friedreich's ataxia by limiting mitochondrial iron accumulation. J. Biol. Chem., 275, 7626–7632.[Abstract/Free Full Text]

  33. Chen, O.S., Hemenway, S. and Kaplan, J. (2002) Inhibition of Fe–S cluster biosynthesis decreases mitochondrial iron export: evidence that Yfh1p affects Fe–S cluster synthesis. Proc. Natl Acad. Sci. USA, 99, 12321–12326.[Abstract/Free Full Text]

  34. Santos, R., Dancis, A., Eide, D., Camadro, J.M. and Lesuisse, E. (2003) Zinc suppresses the iron-accumulation phenotype of Saccharomyces cerevisiae lacking the yeast frataxin homologue (Yfh1). Biochem. J., 375, 247–254.[CrossRef][Web of Science][Medline]

  35. Schena, M., Picard, D. and Yamamoto, K.R. (1991) Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol., 194, 389–398.[Web of Science][Medline]

  36. Branda, S.S., Yang, Z.Y., Chew, A. and Isaya, G. (1999) Mitochondrial intermediate peptidase and the yeast frataxin homolog together maintain mitochondrial iron homeostasis in Saccharomyces cerevisiae. Hum. Mol. Genet., 8, 1099–1110.[Abstract/Free Full Text]

  37. Cavadini, P., Gellera, C., Patel, P.I. and Isaya, G. (2000) Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae. Hum. Mol. Genet., 9, 2523–2530.[Abstract/Free Full Text]

  38. Corson, L.B., Folmer, J., Strain, J.J., Culotta, V.C. and Cleveland, D.W. (1999) Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase. J. Biol. Chem., 274, 27590–27596.[Abstract/Free Full Text]

  39. Chen, C., Dewaele. S., Braeckman, B., Desmyter, L., Verstraelen, J., Borgonie, G., Vanfleteren, J. and Contreras, R. (2003) A high-throughput screening system for genes extending life-span. Exp. Gerontol., 38, 1051–1063.[CrossRef][Web of Science][Medline]

  40. Seo, H.Y., Chung, Y.J., Kim, S.J., Park, C.U. and Kim, K.S. (2003) Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 63, 57–63.[CrossRef][Web of Science][Medline]

  41. Zhao, G., Bou-Abdallah, F., Arosio, P., Levi, S., Janus-Chandler, C. and Chasteen, N.D. (2003) Multiple pathways for mineral core formation in mammalian apoferritin. The role of hydrogen peroxide. Biochemistry, 42, 3142–3150.[CrossRef][Medline]

  42. Lill, R., Diekert, K., Kaut, A., Lange, H., Pelzer, W., Prohl, C. and Kispal, G. (1999) The essential role of mitochondria in the biogenesis of cellular iron–sulfur proteins. Biol. Chem., 380, 1157–1166.[CrossRef][Web of Science][Medline]

  43. Puccio, H., Simon, D., Cossee, M., Criqui-Filipe, P., Tiziano, F., Melki, J., Hindelang, C., Matyas, R., Rustin, P. and Koenig. M. (2001) Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe–S enzyme deficiency followed by intramitochondrial iron deposits. Nat. Genet., 27, 181–186.[CrossRef][Web of Science][Medline]

  44. Simon, D., Seznec, H., Gansmuller, A., Carelle, N., Weber, P., Metzger, D., Rustin, P., Koenig, M. and Puccio, H. (2004) Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia. J. Neurosci., 24, 1987–1995.[Abstract/Free Full Text]

  45. Tan, G., Chen, L.S., Lonnerdal, B., Gellera, C., Taroni, F.A. and Cortopassi, G.A. (2001) Frataxin expression rescues mitochondrial dysfunctions in FRDA cells. Hum. Mol. Genet., 10, 2099–2107.[Abstract/Free Full Text]

  46. Drapier, J.C. and Hibbs, J.B., Jr (1996) Aconitases: a class of metalloproteins highly sensitive to nitric oxide synthesis. Methods Enzymol., 269, 26–36.[Web of Science][Medline]

  47. Munujos, P., Coll-Canti, J., Gonzalez-Sastre, F. and Gella. F.J. (1993) Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor. Anal. Biochem., 212, 506–509.[CrossRef][Web of Science][Medline]

  48. Zeikus, J.G., Fuchs, G., Kenealy, W. and Thauer, R.K. (1977) Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum. J. Bacteriol., 132, 604–613.[Abstract/Free Full Text]

  49. Cairo, G., Ronchi, R., Buccellato, F.R., Veber, D., Santambrogio, P. and Scalabrino, G. (2002) Regulation of the ferritin H subunit by vitamin B12 (cobalamin) in rat spinal cord. J. Neurosci. Res., 69, 117–124.[CrossRef][Web of Science][Medline]


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