Decreased expression of Yfh1p in the budding yeast, Saccharomyces cerevisiae, and the orthologous human gene frataxin results in respiratory deficiency and mitochondrial iron accumulation. The absence of Yfh1p decreases mitochondrial iron export. We demonstrate that decreased expression of Nfs1p, the yeast cysteine desulfurase that plays a central role in Fe-S cluster synthesis, also results in mitochondrial iron accumulation due to decreased export of mitochondrial iron. In the absence of Yfh1p, activity of Fe-Scontaining enzymes (aconitase, succinate dehydrogenase) is decreased, whereas the activity of a non-Fe-S-containing enzyme (malate dehydrogenase) is unaffected. Aconitase protein was abundant even though the activity of aconitase was decreased in both aerobic and anaerobic conditions. These results demonstrate a direct role of Yfh1p in the formation of Fe-S clusters and indicate that mitochondrial iron export requires Fe-S cluster biosynthesis.F rataxin is a highly conserved protein found in all eukaryotes.The protein is encoded by a nuclear gene and is localized in the mitochondrial matrix. Deficits in frataxin protein result in Friedreich ataxia, a neurologic and cardiac disorder (1). Deletion of Yfh1p, the frataxin orthologue in yeast, leads to a respiratory defect resulting from excessive mitochondrial iron accumulation, which increases oxidant damage (2). The increase in mitochondrial iron is caused by malregulation of the mitochondrial iron cycle (3). Mitochondrial iron accumulation is only seen when cytosolic iron levels are high. Reduction in cytosolic iron due to either low-iron media (3, 4), deletion of genes required for high-affinity iron transport (3), or increased vacuolar iron transport (5), prevents excessive mitochondrial iron accumulation and will preserve respiratory activity in ⌬yfh1 cells.In yeast, the proteins involved in Fe-S cluster synthesis are compartmentalized in the mitochondria where Fe-S clusters are synthesized for use both in mitochondria and for export to cytosolic proteins (6). Excessive mitochondrial iron is also seen as a result of deletion of genes required for synthesis of Fe-S clusters (for review, see ref. 6). For example, decreased expression of Nfs1p, the cysteine desulfurase (7, 8), Isu1p, a putative scaffolding protein (9), Yah1p, a putative ferredoxin (10), and Arh1p, a putative ferredoxin reductase (11), results in mitochondrial iron accumulation. In addition, reduced expression of Atm1p, a putative mitochondrial Fe-S exporter, also leads to accumulation of toxic levels of mitochondrial iron (7). The mechanism by which iron accumulates has not been elucidated. Herein, we describe that reduced expression of a critical enzyme involved in Fe-S synthesis, Nfs1p, results in mitochondrial iron accumulation through decreased mitochondrial iron export, similar to that seen in ⌬yfh1 cells (3). This result indicates that mitochondrial iron export depends on Fe-S cluster synthesis, suggesting that Yfh1p may play a role in Fe-S cluster synthesis. To test this hypothesis...

Deletion of the yeast homologue of frataxin, YFH1, results in mitochondrial iron accumulation and respiratory deficiency (petite formation). We used a genetic screen to identify mutants that modify iron-associated defects in respiratory activity in ⌬yfh1 cells. A deletion in the peroxisomal citrate synthase CIT2 in ⌬yfh1 cells decreased the rate of petite formation. Conversely, overexpression of CIT2 in ⌬yfh1 cells increased the rate of respiratory loss. Citrate toxicity in ⌬yfh1 cells was dependent on iron but was independent of mitochondrial respiration. Citrate toxicity was not restricted to iron-laden mitochondria but also occurred when iron accumulated in cytosol because of impaired vacuolar iron storage. These results suggest that high levels of citrate may promote iron-mediated tissue damage.T he yeast YFH1 gene and its mammalian orthologue, frataxin, are nuclear genes that encode mitochondrial proteins (1). In humans, mutations in frataxin are responsible for the neurologic and cardiac disease Friedreich's ataxia (2). The most common mutation of frataxin is an expansion of a GAA triplet within the first intron of the frataxin gene (3). The effect of the triplet expansion is to reduce frataxin transcription and protein concentration. Although the frataxin protein is highly conserved in eukaryotes, there is no consensus regarding the function of Yfh1p͞frataxin. Suggested roles for the protein include mitochondrial iron storage (4, 5), iron-sulfur biosynthesis (2, 6), regulation of respiration (7), and control of antioxidant defenses (8). Although the function of Yfh1p͞frataxin is unknown, it is thought that the pathophysiology of the human disorder results from a mitochondrial defect (9). In yeast, a deficit of Yfh1p leads to accumulation of mitochondrial iron, which reacts with oxygen metabolites to generate oxygen radicals, resulting in a respiratory deficit (1, 10). We used a genetic screen to identify genes that would preserve the respiratory activity of ⌬yfh1 cells. We report that deletion of CIT2, a gene that encodes an extramitochondrial citrate synthase, can markedly attenuate the iron toxicity in ⌬yfh1 cells and preserve respiratory activity. Alternatively, overexpression of CIT2 can induce toxicity when iron accumulates in either the mitochondria or in the cytosol, the latter as a result of impaired iron storage. Although extracellular citrate is an iron chelator, these results suggest that intracellular citrate iron complexes are toxic. Materials and MethodsStrains, Growth Media, and Plasmids. DY150 [MATa,112,] and DY1457 [MAT␣,112, ade6,] were derived from the W303 strain of Saccharomyces cerevisiae. The METYFH1 strain [⌬yfh1, pMET3YFH1[URA3] (a yfh1 strain that has a plasmid containing the YFH1 gene controlled by the MET3 promoter in a vector that has a URA3 gene)] was generated by crossing the ⌬yfh1 [MATa,112,, yfh1::HIS3] strain with DY1457 as described (10). The expression of YFH1 was inhibited by growing cells in complete synthetic media (CM) supplemented with methionine (330 g͞ml).Gene de...