The Yeast Connection to Friedreich Ataxia

Abstract

There have been both genetic and biochemical advances in the understanding of inherited neurodegenerative diseases, such as Huntington disease (HD) and Friedreich ataxia (FA). In FA, these advances have come from the following two approaches: the mapping of the disease gene in humans, and work with a distantly related model organism, Saccharomyces cerevisiae. These two approaches converged, with the study of the human gene leading to the yeast homologue and the study of the yeast mutant phenotypes leading to the human homologue and its associated disease. FA has an estimated prevalence of 1/50,000 in European populations, making it the most common inherited ataxia. The neurologic symptoms, which start during adolescence, include gait and limb ataxia, lower limb areflexia and pyramidal weakness, loss of proprioception, and dysarthria. Most patients develop hypertrophic cardiomyopathy and skeletal abnormalities, and some become diabetic (Durr et al. Durr et al., 1996Durr A Cossee M Agid Y Campuzano V Mignard C Penet C Mandel JL et al.Clinical and genetic abnormalities in patients with Friedreich's ataxia.New Engl J Med. 1996; 335: 1169-1175Crossref PubMed Scopus (812) Google Scholar). These symptoms progress with age, such that most patients become wheelchair-bound by their late twenties and die by their mid-thirties—most commonly of congestive heart failure. Genetically, FA belongs to a class of neurodegenerative disorders in which the underlying gene, FRDA1, carries an unstable trinucleotide-repeat sequence. At least eight other members of this class have been identified, including HD and many types of spinocerebellar ataxia. However, key genetic features separate FA from the other trinucleotide-repeat disorders. First, the sequence of the trinucleotide repeat in the FRDA1 gene is GAA (Campuzano et al. Campuzano et al., 1996Campuzano V Montermini L Molto MD Pianese L Cossee M Cavalcanti F Monros E et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2145) Google Scholar), whereas a CAG repeat occurs in the other trinucleotide-associated ataxias, and other repeats (CTG or CGG) are seen in other trinucleotide diseases. Second, the GAA repeat of FRDA1 is located in the first intron and is therefore noncoding, whereas the CAG repeat in HD and the spinocerebellar ataxias always occurs within an exon and encodes glutamine (Campuzano et al. Campuzano et al., 1996Campuzano V Montermini L Molto MD Pianese L Cossee M Cavalcanti F Monros E et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2145) Google Scholar). The third difference is that FA is inherited in a recessive manner, and multiple lines of evidence suggest that loss of function leads to the disease. In contrast, in the other trinucleotide-repeat disorders, whether the repeat occurs in an expressed DNA sequence (Paulson PaulsonPaulson HL. Protein fate in neurodegenerative proteinopathies: polyglutamine diseases join the (mis)fold. Am J Hum Genet 64:339–345 (in this issue)Google Scholar [in this issue]) or in a 3′ untranslated sequence (Timchenko TimchenkoTimchenko LT. Myotonic distrophy: the role of RNA CUG triplet repeats. Am J Hum Genet 64:360-364. (in this issue)Google Scholar [in this issue]), the mutation is inherited in a dominant manner, and it is a gain of function of the affected protein or RNA that perturbs cell physiology. The severity of the disease correlates with decreased FRDA1 expression and with the length of the hyperexpansive repeat. Normally, this gene, which encodes the protein frataxin, contains <39 GAA repeats, but in patients with FA, this locus contains 66–1,700 repeat units. This hyperexpansion results in marked decreases in frataxin mRNA levels, thought to result from the formation of an unusual non-β DNA structure inhibiting transcription (Bidichandani et al. Bidichandani et al., 1998Bidichandani SI Ashizawa T Patel PI The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure.Am J Hum Genet. 1998; 62: 111-121Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar; Sinden SindenSinden RR. Biological implications of the DNA structures associated with disease-causing triplet repeats. Am J Hum Genet 64:346-353. (in this issue)Google Scholar [in this issue]). More than 95% of patients with FA are homozygous for the GAA hyperexpansion, although the alleles are polymorphic in the number of GAA repeats. Studies have shown a correlation between the length of the GAA expansion on the smaller allele and severity of disease (Durr et al. Durr et al., 1996Durr A Cossee M Agid Y Campuzano V Mignard C Penet C Mandel JL et al.Clinical and genetic abnormalities in patients with Friedreich's ataxia.New Engl J Med. 1996; 335: 1169-1175Crossref PubMed Scopus (812) Google Scholar). An inverse correlation between GAA expansion size and frataxin protein levels has been observed in lymphoblast cell lines from patients with FA (Campuzano et al. Campuzano et al., 1996Campuzano V Montermini L Molto MD Pianese L Cossee M Cavalcanti F Monros E et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2145) Google Scholar). Together, these findings suggest that lack of frataxin protein in critical tissues leads to FA. The remaining 5% of patients with FA are compound heterozygotes for the GAA expansion on one allele and carry point mutations within FRDA1 on the other allele. The most common disease-causing point mutation in frataxin is I154F (numbering based on the initiator methionine of the predicted open reading frame [ORF]), prevalent in some southern Italian families. Those individuals carrying this missense mutation on one allele, together with the hyperexpansion on the other allele, are indistinguishable in disease severity when compared with homozygous relatives who carry the GAA triplet expansion on both alleles (Campuzano et al. Campuzano et al., 1996Campuzano V Montermini L Molto MD Pianese L Cossee M Cavalcanti F Monros E et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2145) Google Scholar). Another missense mutation in frataxin, G130V, compounded with a hyperexpansive allele, is associated with a milder and more slowly progressive disease course (Bidichandani et al. Bidichandani et al., 1997Bidichandani SI Ashizawa T Patel PI Atypical Friedreich ataxia caused by compound heterozygosity for a novel missense mutation and the GAA triplet-repeat expansion.Am J Hum Genet. 1997; 60: 1251-1256PubMed Google Scholar). Of note is that disease-causing point mutations in frataxin occur in residues conserved with the homologous yeast protein. The precise biochemical function that is disrupted is unknown, but it may relate to iron metabolism. Ironclad EvidenceIn yeast cells, as in human cells, nuclear and mitochondrial genes are required, for mitochondria to function. Yeast mtDNA encode eight polypeptides (compared with 13 open reading frames in human mtDNA), plus ribosomal and transfer RNA. The major mitochondrial functions, oxidative phosphorylation and oxidative metabolism of fatty acids, clearly are essential in metazoans, but yeast cells completely lacking mtDNA can be grown indefinitely, so long as fermentable carbon sources, such as glucose, are provided in their medium. For this reason, nuclear or mitochondrial mutations that affect oxidative phosphorylation can readily be identified as mutant yeast cells that fail to grow on a nonfermentable carbon source, such as glycerol. Such yeast strains are termed “pet” mutants, because their growth, even on glucose, is slow, leading to small (“petite”) colonies. When pet mutants arise from mutations in mtDNA, they may be described as either ρ−or ρo, depending on whether the mtDNA has undergone large deletions or has been lost from the cell entirely. The figure below depicts the strategy we applied to show that one pet yeast strain, mutant for a nucleus-encoded gene, Yfh1, is ρ−or ρo as a consequence of this mutation. Yfh1 encodes a homologue of the human frataxin protein, which is implicated in Friedreich ataxia. We crossed the yfh1 mutant with a ρo strain, in which the nuclear genome was wild type. The resulting diploid yeast cell, like the yfh1− parental strain, could not grow on the glycerol carbon source, indicating that mtDNA in yfh1− cannot restore mitochondrial function, even when the Yfh1p protein is available from the other parental strain.A link between iron and mtDNA damage was suggested, from the independent cloning of YFH1 as a high-copy suppressor of a mutant yeast that was defective in its use of intracellular iron. Loss of YFH1 function leads to mtDNA damage and to mitochondrial accumulation of iron. Another relevant gene, SSQ1, was identified in a screen for yeast mutants exhibiting altered iron homeostasis. Like yfh1 mutants, ssq1 cells accumulate iron in mitochondria and sustain mitochondrial DNA damage. Because SSQ1 encodes a putative mitochondrial Hsp70, we looked for evidence that chaperone defects affect Yfh1p maturation, and we observed that Yfh1p processing in mitochondria was compromised in ssq1 yeast. We suggest that the human frataxin protein plays a similar role in intracellular iron trafficking and that other human genes, analogous to SSQ1, may be relevant to the pathogenesis of certain other disorders related to Friedreich ataxia. In yeast cells, as in human cells, nuclear and mitochondrial genes are required, for mitochondria to function. Yeast mtDNA encode eight polypeptides (compared with 13 open reading frames in human mtDNA), plus ribosomal and transfer RNA. The major mitochondrial functions, oxidative phosphorylation and oxidative metabolism of fatty acids, clearly are essential in metazoans, but yeast cells completely lacking mtDNA can be grown indefinitely, so long as fermentable carbon sources, such as glucose, are provided in their medium. For this reason, nuclear or mitochondrial mutations that affect oxidative phosphorylation can readily be identified as mutant yeast cells that fail to grow on a nonfermentable carbon source, such as glycerol. Such yeast strains are termed “pet” mutants, because their growth, even on glucose, is slow, leading to small (“petite”) colonies. When pet mutants arise from mutations in mtDNA, they may be described as either ρ−or ρo, depending on whether the mtDNA has undergone large deletions or has been lost from the cell entirely. The figure below depicts the strategy we applied to show that one pet yeast strain, mutant for a nucleus-encoded gene, Yfh1, is ρ−or ρo as a consequence of this mutation. Yfh1 encodes a homologue of the human frataxin protein, which is implicated in Friedreich ataxia. We crossed the yfh1 mutant with a ρo strain, in which the nuclear genome was wild type. The resulting diploid yeast cell, like the yfh1− parental strain, could not grow on the glycerol carbon source, indicating that mtDNA in yfh1− cannot restore mitochondrial function, even when the Yfh1p protein is available from the other parental strain. A link between iron and mtDNA damage was suggested, from the independent cloning of YFH1 as a high-copy suppressor of a mutant yeast that was defective in its use of intracellular iron. Loss of YFH1 function leads to mtDNA damage and to mitochondrial accumulation of iron. Another relevant gene, SSQ1, was identified in a screen for yeast mutants exhibiting altered iron homeostasis. Like yfh1 mutants, ssq1 cells accumulate iron in mitochondria and sustain mitochondrial DNA damage. Because SSQ1 encodes a putative mitochondrial Hsp70, we looked for evidence that chaperone defects affect Yfh1p maturation, and we observed that Yfh1p processing in mitochondria was compromised in ssq1 yeast. We suggest that the human frataxin protein plays a similar role in intracellular iron trafficking and that other human genes, analogous to SSQ1, may be relevant to the pathogenesis of certain other disorders related to Friedreich ataxia. Phenotypic variants of FA, which nonetheless are caused by mutations at the FRDA1 locus, include FA with retained lower limb reflexes, late-onset FA, and the more slowly progressing Acadian FA variant (Palau et al. Palau et al., 1995Palau F De Michele G Vilchez JJ Pandolfo M Monros E Cocozza S Smeyers P et al.Early-onset ataxia with cardiomyopathy and retained tendon reflexes maps to the Friedreich's ataxia locus on chromosome 9q.Ann Neurol. 1995; 37: 359-362Crossref PubMed Scopus (82) Google Scholar). These less-severe variants of FA often occur in individuals carrying intermediate lengths of the GAA repeat. Because of this proportionate loss of expression of FRDA1 as the GAA trinucleotide expands, the disease mechanism in FA is more analogous to that of fragile X–linked mental retardation (FXMR) than to the other repeat-associated ataxias. However, the analogy to FXMR is also imperfect, because, in that disorder, loss of gene expression is associated with CpG methylation in long CGG tracts in the 5′ untranslated region of the first exon of FMR1. Such a mechanism is incompatible with the structure of the intronic GAA repeats in FRDA1. At the time of its identification, the deduced 210–amino acid sequence of frataxin revealed no clue to its function, other than the presence of an N-terminal sequence that suggested mitochondrial localization (Campuzano et al. Campuzano et al., 1996Campuzano V Montermini L Molto MD Pianese L Cossee M Cavalcanti F Monros E et al.Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2145) Google Scholar). The frataxin ORF is homologous to ORFs from Caenorhabditis elegans and S. cerevisiae. A homologue is also present in a gram-negative bacterial species, whose genomic structure suggests that it is related to the predecessors of eukaryotic mitochondria. Thus, the frataxin gene may have existed first within the ancestral mtDNA of eukaryotes and then moved to the nuclear genome at some time early in eukaryotic evolution. The connection of FA with mitochondrial function was significantly strengthened by the study of the yeast frataxin homologue encoded by YFH1. The encoded protein was localized to yeast mitochondria by immunofluorescence microscopy. Furthermore, disruption of this gene caused the yeast to be unable to grow on nonfermentable carbon sources (e.g., glycerol plus ethanol) and to grow poorly on fermentable carbon sources (e.g., glucose). The yfh1 mutant strains thus display a “petite” phenotype (see sidebar) and are unable to carry out normal oxidative phosphorylation (Babcock et al. Babcock et al., 1997Babcock M de Silva D Oaks R Davis-Kaplan S Jiralerspong S Montermini L Pandolfo M et al.Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin.Science. 1997; 276: 1709-1712Crossref PubMed Scopus (780) Google Scholar; Foury and Cazzalini Foury and Cazzalini, 1997Foury F Cazzalini O Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria.FEBS Lett. 1997; 411: 373-377Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar; Koutnikova et al. Koutnikova et al., 1997Koutnikova H Campuzano V Foury F Dolle P Cazzalini O Koenig M Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.Nat Genet. 1997; 16: 345-351Crossref PubMed Scopus (407) Google Scholar; Wilson and Roof Wilson and Roof, 1997Wilson RB Roof DM Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue.Nat Genet. 1997; 16: 352-357Crossref PubMed Scopus (315) Google Scholar), at least in part because of mtDNA damage. A major insight into FA came when seemingly unrelated studies on metal metabolism in yeast suggested the involvement of iron. Iron transport into yeast is mediated by ferric reductases of the plasma membrane (Fre1p and Fre2p), which reduce iron to its ferrous form before it is brought into the cell through Ftr1p, an iron permease, and its associated multicopper oxidase, Fet3p (Askwith and Kaplan Askwith and Kaplan, 1998Askwith C Kaplan J Iron and copper transport in yeast and its relevance to human disease.Trends Biochem Sci. 1998; 23: 135-138Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). All of these genes are under the control of Aft1p, an iron-sensitive transcriptional factor. When yeast lack iron, Aft1p activates transcription of the iron-uptake components, and conversely, when iron is plentiful, Aft1p-dependent transcription ceases. Although this cellular import pathway is well understood, little is known about the trafficking of iron once it enters the cell. Therefore, Li and Kaplan (Li and Kaplan, 1996Li L Kaplan J Characterization of yeast methyl sterol oxidase (ERG25) and identification of a human homologue.J Biol Chem. 1996; 271: 16927-16933Crossref PubMed Scopus (85) Google Scholar) sought mutants that were defective in intracellular iron usage. YFH1 was identified as a high copy-number suppressor of one such mutant, thereby linking the yeast frataxin homologue to iron (Babcock et al. Babcock et al., 1997Babcock M de Silva D Oaks R Davis-Kaplan S Jiralerspong S Montermini L Pandolfo M et al.Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin.Science. 1997; 276: 1709-1712Crossref PubMed Scopus (780) Google Scholar). Cells lacking YFH1 exhibit constitutive activity of the high-affinity iron uptake system. Cytosolic iron is diminished in yfh1 cells (Knight et al. Knight et al., 1998Knight SAB Sepuri NBV Pain D Dancis A Mt-Hsp70 Homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis.J Biol Chem. 1998; 273: 18389-18393Crossref PubMed Scopus (147) Google Scholar), whereas mitochondrial iron is ≥10-fold higher (Babcock et al. Babcock et al., 1997Babcock M de Silva D Oaks R Davis-Kaplan S Jiralerspong S Montermini L Pandolfo M et al.Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin.Science. 1997; 276: 1709-1712Crossref PubMed Scopus (780) Google Scholar; Foury and Cazzalini Foury and Cazzalini, 1997Foury F Cazzalini O Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria.FEBS Lett. 1997; 411: 373-377Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). This suggests that lack of Yfh1p causes iron accumulation in mitochondria and breakdown of cellular iron homeostasis. The exact cause of the mitochondrial iron accumulation is not known; however, a role for Yfh1p in efflux of iron from the mitochondria has been suggested (Askwith and Kaplan Askwith and Kaplan, 1998Askwith C Kaplan J Iron and copper transport in yeast and its relevance to human disease.Trends Biochem Sci. 1998; 23: 135-138Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). With new insight gained from yeast genetic studies that suggest FA may be associated with altered cellular iron distrubution, it is possible, in hindsight, to find supportive evidence from earlier studies in the biochemical and clinical literature. One such study evaluated the fate of radioactive iron citrate administered to patients with FA, compared with individuals with Hallervorden-Spatz disease (Szanto and Gallyas Szanto and Gallyas, 1966Szanto J Gallyas F A study of iron metabolism in neuropsychiatric patients. Hallervorden-Spatz disease.Arch Neurol. 1966; 14: 438-442Crossref PubMed Scopus (42) Google Scholar). The patients with FA exhibited more-rapid turnover of iron in plasma and in red blood cells, suggesting a decrease in red-cell survival. Plasma iron concentration, plasma iron binding capacity, and urinary iron were not affected. Measurements of levels of hemoglobin, bilirubin, lactate dehydrogenase, and reticulocyte counts were not reported. A subsequent study has confirmed that patients with FA exhibit normal levels of serum iron and ferritin (Wilson et al. Wilson et al., 1998Wilson RB Lynch DR Fischbeck KH Normal serum iron and ferritin concentrations in patients with Friedreich's ataxia.Ann Neurol. 1998; 44: 132-134Crossref PubMed Scopus (46) Google Scholar). However, these measurements reflect red-cell iron metabolism, which may be regulated by specifically dedicated feedback loops, as compared with other tissues. Despite the ubiquitous expression of frataxin in all tissues, the primary effects of FA are seen in the central nervous system, specifically in large sensory neurons and dorsal root ganglia. The heart is also targeted, and the resulting cardiomyopathy limits life expectancy (Koutnikova et al. Koutnikova et al., 1997Koutnikova H Campuzano V Foury F Dolle P Cazzalini O Koenig M Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.Nat Genet. 1997; 16: 345-351Crossref PubMed Scopus (407) Google Scholar). The reasons for this pattern of tissue sensitivity are not known, although they may relate to the metabolic rate and abundance of mitochondria in these tissues (Koutnikova et al. Koutnikova et al., 1997Koutnikova H Campuzano V Foury F Dolle P Cazzalini O Koenig M Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.Nat Genet. 1997; 16: 345-351Crossref PubMed Scopus (407) Google Scholar). Postmortem examination of hearts from patients with FA have shown zones of cardiac muscle degeneration, with many iron deposits within myocardial fibers (Lamarche et al. Lamarche et al., 1980Lamarche JB Cote M Lemieux B The cardiomyopathy of Friedreich's ataxia morphological observations in 3 cases.Can J Neurol Sci. 1980; 7: 389-396Crossref PubMed Scopus (161) Google Scholar). Electron microscopy of myocardial fibers showed electron-dense deposits, surrounded by membranes and associated with damaged mitochondria. These deposits were termed “lipofuscin”—an ill-defined autofluorescent aggregate, containing polyunsaturated lipids and proteins. Although these deposits were not analyzed for iron, it is tempting to speculate that it might be present. Iron is a cofactor for numerous heme and Fe-S proteins of mitochondria. In the electron transport chains of yeast and mammals, for example, complex II (succinate: ubiquinone oxidoreductase) contains Fe-S clusters as prosthetic groups, whereas complex III (bc1 complex) contains both Fe-S in the Rieske iron-sulfur protein and heme in the form of cytochromes b and c1, and complex IV (cytochrome c oxidase) contains heme as cytochromes a and a3. Also present in mitochondria is aconitase, an Fe-S enzyme of the tricarboxylic acid cycle. In endomyocardial biopsied tissue from two patients with FA, activity levels of complexes II and III were found to be in the low to normal range. Of the enzymes analyzed, aconitase activity exhibited a marked decrease, relative to that of normal controls. Low enzyme activities were noted only in the diseased heart tissue and not in the unaffected tissues that included skeletal muscle, lymphocytes, or skin fibroblasts (Rotig et al. Rotig et al., 1997Rotig A de Lonlay P Chretien D Foury F Koenig M Sidi D Munnich A et al.Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia.Nat Genet. 1997; 17: 215-217Crossref PubMed Scopus (837) Google Scholar). Similarly, in the yfh1 yeast mutants, the activities of complexes II, III, and IV, as well as aconitase, were reduced. Overall, the yfh1 yeast mutant and disease tissue from patients with FA exhibit extraordinary similarities. Iron accumulates in mitochondria (in the yeast mutant and perhaps in the human disease tissue) in vast excess, and yet, paradoxically, iron proteins are deficient. The effects are more severe in the yeast than in the FA tissue, perhaps reflecting the difference between reduced expression of FRDA1 in humans versus YFH1 deletion in yeast. In a screen for mutations affecting iron metabolism in yeast, another mutant that accumulates iron in the mitochondria was isolated (Knight et al. Knight et al., 1998Knight SAB Sepuri NBV Pain D Dancis A Mt-Hsp70 Homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis.J Biol Chem. 1998; 273: 18389-18393Crossref PubMed Scopus (147) Google Scholar). The defect in this case was in a nuclear gene encoding a mitochondrial heat-shock protein 70 (Hsp70). In yeast, there are two mitochondrial Hsp70 (homologous to DnaK in bacteria), encoded by the genes SSC1 and SSQ1. These proteins belong to the class of Hsp70 chaperones that facilitate the importing, processing, and folding of proteins in various organelles. Most proteins destined for the mitochondria are encoded in the nucleus and translated on cytoplasmic ribosomes. Hydrophobic regions of proteins interact with Ssc1p in the mitochondrial matrix during import (Kang et al. Kang et al., 1990Kang PJ Ostermann J Shilling J Neupert W Craig EA Pfanner N Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins.Nature. 1990; 348: 137-143Crossref PubMed Scopus (526) Google Scholar). This chaperone acts in association with at least 2 other proteins, Mdj1p (a homologue of the bacterial protein DnaJ) and Mge1p (a GrpE homologue). These three proteins use energy from ATP hydrolysis, to ensure that the incoming protein folds correctly (Horst et al. Horst et al., 1997Horst M Oppliger W Rospert S Schönfeld H-J Schatz G Azem A Sequential action of two hsp70 complexes during protein import into mitochondria.EMBO J. 1997; 16: 1842-1849Crossref PubMed Scopus (106) Google Scholar). For a subset of proteins, a second maturation system is required, which consists of Hsp60 (GroEL) and Hsp10 (GroES). This complex traps the nonfolded protein and allows the protein to fold into its native form. The protein is then released, after ATP hydrolysis. The released protein may achieve its native conformation immediately, or it may require additional rounds of interaction with the chaperone before reaching its final state (Martin Martin, 1997Martin J Molecular chaperones and mitochondrial protein folding.J Bioenerg Biomembr. 1997; 29: 35-43Crossref PubMed Scopus (59) Google Scholar). The Hsp70 gene, SSC1, is essential and abundantly expressed in yeast. The second mitochondrial Hsp70, SSQ1, originally was cloned as a homologue of SSC1 (Schilke et al. Schilke et al., 1996Schilke B Forster J Davis J James P Walter W Laloraya S Johnson J et al.The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.J Cell Biol. 1996; 134: 603-613Crossref PubMed Scopus (70) Google Scholar). SSQ1 was found to be nonessential and expressed at low levels, when compared with SSC1. It was also found to play a role in maintaining mtDNA (Schilke et al. Schilke et al., 1996Schilke B Forster J Davis J James P Walter W Laloraya S Johnson J et al.The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.J Cell Biol. 1996; 134: 603-613Crossref PubMed Scopus (70) Google Scholar). SSQ1 was independently cloned from a screen of yeast mutants with defective cellular iron homeostasis (Knight et al. Knight et al., 1998Knight SAB Sepuri NBV Pain D Dancis A Mt-Hsp70 Homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis.J Biol Chem. 1998; 273: 18389-18393Crossref PubMed Scopus (147) Google Scholar). Like yfh1 mutants, ssq1 cells exhibit increased cellular iron uptake, iron diversion to the mitochondria, and accumulation of mtDNA damage (Knight et al. Knight et al., 1998Knight SAB Sepuri NBV Pain D Dancis A Mt-Hsp70 Homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis.J Biol Chem. 1998; 273: 18389-18393Crossref PubMed Scopus (147) Google Scholar). This phenotypic similarity between yfh1 and ssq1 mutations prompted investigations to determine whether Ssq1p is required for Yfh1p import and processing in mitochondria. In wild-type cells, Yfh1p is imported as a preprotein and is subjected to two sequential processing cleavages at the N-terminus (Knight et al. Knight et al., 1998Knight SAB Sepuri NBV Pain D Dancis A Mt-Hsp70 Homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis.J Biol Chem. 1998; 273: 18389-18393Crossref PubMed Scopus (147) Google Scholar). The first cleavage removes ∼2 kDa, and the second cleavage removes another 4 kDa. In the ssq1 mutant, the second cleavage is impaired kinetically. The mechanism by which Ssq1p participates in the second processing step of Yfh1p is not known, but it may hold Yfh1p in a conformation that favors processing. Alternatively, Ssq1p may be required for the correct folding of Yfh1p, for its insertion into a multiprotein complex. SSQ1 appears to be involved in the processing of Yfh1p within mitochondria. By contrast, import and proteolytic processing of Rieske iron-sulfur protein, cytochrome b2, and cytochrome c1, all components of Complex III, do not require Ssq1p. The import and processing of a noniron protein, prePut2, an enzyme of proline biosynthesis, also proceeds normally in the ssq1 mutant strain (Schilke et al. Schilke et al., 1996Schilke B Forster J Davis J James P Walter W Laloraya S Johnson J et al.The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.J Cell Biol. 1996; 134: 603-613Crossr