Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are

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... ubject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis. Recent studies have shown that the yeast frataxin homologue (YFH1, gene; Yfh1p, polypeptide) is a nuclear-encoded mitochondrial protein (1-4) and that its deficiency results in mitochondrial iron overload (1, 2, 5), which in turn leads to increased production of free radicals and loss of mitochondrial function (1). Similarly, iron deposits (6), multiple mitochondrial enzyme deficiencies (7) , and hypersensitivity to oxidative stress (8) have been reported in studies on Friedreich's ataxia (FRDA), 1 a recessively inherited neurodegenerative disease caused by a deficiency of human frataxin (9, 10). Thus, it is believed that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity and that this function is conserved between yeast and mammals (7, 11). Not surprisingly, Yfh1p and mammalian frataxin share similar pathways of mitochondrial import and processing. The Yfh1p precursor (pYfh1p) is imported by isolated yeast mitochondria and processed to an intermediate (iYfh1p) and a mature size (mYfh1p) form (12) . Production of mYfh1p is impaired in mitochondria isolated from yeast with mutations in the mitochondrial Hsp70 homologue Ssq1p, and similar to Yfh1p-deficient yeast (yfh1⌬) (1, 2, 5), ssq1 mutants accumulate large amounts of mitochondrial iron (12), indicating that production of mYfh1p is required for mitochondrial iron homeostasis. The mouse frataxin precursor is also cleaved twice, and missense mutations corresponding to those found in FRDA patients dramatically reduce the efficiency of the second cleavage (13), further demonstrating the importance of proteolytic processing for frataxin function. Mitochondrial processing peptidase (MPP; EC 3.4.24.64) (14) was shown to catalyze conversion of the mouse frataxin precursor to intermediate form, but the peptidase responsible for formation of mature frataxin was not identified (13). Additionally, it has not yet been established whether the intermediate forms of Yfh1p and frataxin represent productive intermediates, in that they are actually processed to the mature form. In this study, we analyze proteolytic processing of Yfh1p and human frataxin and demonstrate that both proteins are processed to the mature form in two sequential steps by MPP. EXPERIMENTAL PROCEDURES Yeast Strains, Plasmids, and Media-The strains used in this study are all isogenic derivatives of strain YPH501 (see Table I ). Construction of oct1⌬, yfh1⌬, and isogenic 0 strains was described previously (15, 16) . For complementation of yfh1⌬ by Yfh1p-myc, a polymerase chain reaction fragment encoding the Yfh1p C terminus fused in-frame to the 9E10 c-myc epitope was synthesized using a sense oligonucleotide complementary to the YFH1 coding sequence upstream of a unique AccI site and an antisense oligonucleotide specifying the 3Ј-end of the YFH1 coding sequence, the 9E10 c-myc epitope coding sequence, a stop codon, 22 base pairs of the YFH1 3Ј-flanking DNA, and a BamHI site. This polymerase chain reaction product was substituted for the 3Ј-region of the YFH1 gene by digestion with AccI and BamHI, yielding a YFH1-myc fusion construct. A centromeric TRP1-based YCplac22-YFH1-myc plasmid was then used to transform the yfh1⌬[YFH1] strain (16) and replace the URA3-based YCp50-YFH1 plasmid, which was eliminated by counterselection with 5-fluoroorotic acid, yielding the yfh1⌬[YFH1myc] strain. Mitochondrial Fractionation-The yfh1⌬[YFH1-myc] strain was grown in SSGD (6.7% bacto-yeast nitrogen base without amino acids, 0.3% yeast extract, 2% galactose, and 0.05% dextrose, supplemented with amino acids and other growth requirements) at 30°C to an A 600 of ϳ2, spheroplasts were prepared and homogenized, and the nuclear (1,000 ϫ g pellet), heavy (3,000 ϫ g pellet), and light (17,000 ϫ g pellet) mitochondrial fractions were separated by differential centrifugation.