The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins

# 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No. 17 2025–2036 The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins Ulrich Mühlenhoff1, Nadine Richhardt1, Michael Ristow2, Gyula Kispal3 and Roland Lill1,* 1 Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch Str. 5, 35033 Marburg, Germany, 2Deutsches Institut für Ernährungsforschung, Arthur-Scheunert-Allee 114, 14558 Potsdam, Germany and 3Institute of Biochemistry, Medical Faculty, University of Pecs, Szigeti ut 12, 7624 Pecs, Hungary Received April 26, 2002; Revised June 20, 2002; Accepted June 21, 2002 INTRODUCTION Friedreich’s ataxia (FRDA) is the most common inherited ataxia and is transmitted in an autosomal recessive fashion (for recent reviews see 1–4). The disease is characterized by degeneration of the large sensory neurons and spinocerebellar tracts. Characteristic symptoms include ataxia of the limbs, areflexia, muscle weakness and skeletal deformities, cardiomyopathy, sensory loss, and increased incidence of diabetes mellitus. FRDA is caused by severely reduced levels of frataxin, a mitochondrial matrix protein (5,6). Deletion of the frataxin gene in mice leads to early embryonic lethality, demonstrating an important role of the protein during embryonic development (7). Histological and biochemical analyses of tissues from FRDA patients and conditional mouse mutants have shown that frataxin defects result in the accumulation of iron deposits within mitochondria (8,9). Homologs of frataxin have been identified in most eukaryotes, from yeasts to humans. Distant homologs are found in many proteobacteria, but the protein has so far not been detected in other bacterial lineages and is absent in archaea (10–14). Its three-dimensional structure does not resemble any known protein- or cofactor-binding motif (15–17); however, frataxin shares weak sequence homology with bacterial proteins involved in tellurite resistance (18). The most intensely studied member of the frataxin family is the yeast frataxin homolog Yfh1p. Deletion of the YFH1 gene (yielding Dyfh1 cells) leads to growth defects on non-fermentable carbon sources, a tendency to lose the mitochondrial DNA (mtDNA), sensitivity to oxidative agents, and iron accumulation within mitochondria (10–12). Depending on the strain background, the severity of these phenotypes differs quite substantially, thus rendering conclusions on the function of frataxin rather difficult. Several different functions have been proposed for mitochondrial frataxin. Most of these proposals are centered around the influence of frataxin on cellular iron homeostasis. Genome-wide analysis of gene expression in yeasts has shown that frataxin deficiency results in enhanced expression of genes that are under the control of the iron-sensing transcription factors AFT1/AFT2, including genes involved in iron uptake (19). Since the AFT regulatory system is induced by low cytosolic iron concentration; (20–22), the deletion of frataxin appears to result in a redistribution of iron from the yeast cytosol to mitochondria. Mitochondrial iron accumulation was explained by a function of *To whom correspondence should be addressed. Tel: þ49 64212866449; Fax: þ49 64212866414; Email: Downloaded from http://hmg.oxfordjournals.org/ at Clarkson University on March 12, 2016 The mitochondrial matrix protein frataxin is depleted in patients with Friedreich’s ataxia, the most common autosomal recessive ataxia. While frataxin is important for intracellular iron homeostasis, its exact cellular role is unknown. Deletion of the yeast frataxin homolog YFH1 yields mutants (Dyfh1) that, depending on the genetic background, display various degrees of phenotypic defects. This renders it difficult to distinguish primary (early) from secondary (late) consequences of Yfh1p deficiency. We have constructed a yeast strain (Gal-YFH1) that carries the YFH1 gene under the control of a galactose-regulated promoter. Yfh1p-deficient Gal-YFH1 cells are far less sensitive to oxidative stress than Dyfh1 mutants, maintain mitochondrial DNA, and synthesize heme at wild-type rates. Yfh1p depletion causes a strong reduction in the assembly of mitochondrial Fe/S proteins both in vivo and in detergent extracts of mitochondria. Impaired Fe/S protein biogenesis explains the respiratory deficiency of Gal-YFH1 cells. Furthermore, Yfh1p-depleted Gal-YFH1 cells show decreased maturation of cytosolic Fe/S proteins and accumulation of mitochondrial iron. This latter phenotype is common for defects in cytosolic Fe/S protein assembly. Together, our data demonstrate a specific role of frataxin in the biosynthesis of cellular Fe/S proteins and exclude most of the previously suggested functions. Friedreich’s ataxia may therefore represent a disorder caused by defects in Fe/S protein maturation. 2026 Human Molecular Genetics, 2002, Vol. 11, No. 17 RESULTS The yeast frataxin homolog Yfh1p is required for respiratory activity of yeast We constructed a conditional Saccharomyces cerevisiae mutant termed Gal-YFH1 in which the 50 -upstream sequence of the YFH1 gene is replaced by the galactose-regulated GAL1-10 promoter using a PCR-mediated DNA replacement procedure (see Materials and Methods). Promoter exchange resulted in the moderate (3-fold) overexpression of Yfh1p when GalYFH1 cells were grown in the presence of galactose (Fig. 1A). Upon cultivation in the absence of galactose, YFH1 expression declined to levels hardly detectable by immunostaining analysis. Yfh1p-depleted Gal-YFH1 cells exhibited wild-type growth on glucose-containing rich media (Fig. 1B). On minimal media supplemented with glucose, cells gave rise to small colonies, whereas hardly any growth was detectable in the presence of glycerol, a non-fermentable carbon source. Thus, the Yfh1p-depleted cells show a behavior typical of a petite phenotype, which is indicative for respiratory deficiency in yeast (31). As judged by DNA staining with DAPI, the cells did not loose the mtDNA, even after extended growth under depleting conditions (not shown). In addition, when the levels of Yfh1p were elevated by shift to minimal medium with galactose, wild-type growth of Gal-YFH1 cells was restored, demonstrating the reversible character of the Yfh1p deficiency (not shown). This feature distinguished these cells from some of the deletion mutant strains Dyfh1 (11,12) and suggested that the loss of mtDNA is an indirect consequence of frataxin deletion. Interestingly, growth of Gal-YFH1 cells on glucosecontaining minimal media could be partially improved by supplementation with glutamate and methionine (not shown). This auxotrophic behavior is reminiscent of mutants impaired in amino acid biosynthetic pathways which involve enzymes with Fe/S clusters (see below). To further demonstrate the specificity of our Gal-YFH1 mutant, we transformed these cells with plasmids containing the yeast (no (...truncated)