Yeast and Human Frataxin Are Processed to Mature Form in Two Sequential Steps by the Mitochondrial Processing Peptidase
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22763
ISSN1083-351X
AutoresSteven S. Branda, Patrizia Cavadini, Jiří Adamec, František Kalousek, Franco Taroni, Grazia Isaya,
Tópico(s)Neurological disorders and treatments
ResumoFrataxin 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 subject 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. 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 subject 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 (1Babcock M. De Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar, 2Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (347) Google Scholar, 3Wilson R.B. Roof D.M. Nat. Genet. 1997; 16: 352-357Crossref PubMed Scopus (326) Google Scholar, 4Koutnikova H. Campuzano V. Foury F. Dollé P. Cazzalini O. Koenig M. Nat. Genet. 1997; 16: 345-351Crossref PubMed Scopus (430) Google Scholar) and that its deficiency results in mitochondrial iron overload (1Babcock M. De Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar, 2Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (347) Google Scholar, 5Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), which in turn leads to increased production of free radicals and loss of mitochondrial function (1Babcock M. De Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar). Similarly, iron deposits (6Lamarche J.B. Côté M. Lemieux B. J. Can. Sci. Neurol. 1980; 7: 389-396Crossref PubMed Scopus (173) Google Scholar), multiple mitochondrial enzyme deficiencies (7Rötig A. De Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genet. 1997; 17: 215-217Crossref PubMed Scopus (880) Google Scholar), and hypersensitivity to oxidative stress (8Wong A. Yang J. Cavadini P. Gellera C. Lonnerdal B. Taroni F. Cortopassi G. Hum. Mol. Genet. 1999; 8: 425-430Crossref PubMed Scopus (326) Google Scholar) have been reported in studies on Friedreich's ataxia (FRDA), 1The abbreviations used are: FRDA, Friedreich's ataxia; MPP, mitochondrial processing peptidase: PAGE, polyacrylamide gel electrophoresis; IMP, inner membrane peptidase; MIP, mitochondrial intermediate peptidase; Cyt b 2, cytochromeb 2 a recessively inherited neurodegenerative disease caused by a deficiency of human frataxin (9Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monrós E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2308) Google Scholar, 10Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (616) Google Scholar). 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 (7Rötig A. De Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genet. 1997; 17: 215-217Crossref PubMed Scopus (880) Google Scholar, 11Pandolfo M. Neuromusc. Dis. 1998; 8: 409-415Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (12Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Production of mYfh1p is impaired in mitochondria isolated from yeast with mutations in the mitochondrial Hsp70 homologue Ssq1p, and similar to Yfh1p-deficient yeast (yfh1Δ) (1Babcock M. De Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar, 2Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (347) Google Scholar, 5Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), ssq1mutants accumulate large amounts of mitochondrial iron (12Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), 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 (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar), further demonstrating the importance of proteolytic processing for frataxin function. Mitochondrial processing peptidase (MPP; EC 3.4.24.64) (14Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (978) Google Scholar) 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 (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar). 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. The strains used in this study are all isogenic derivatives of strain YPH501 (see Table I). Construction of oct1Δ, yfh1Δ, and isogenic ρ0 strains was described previously (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar, 16Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholar). For complementation of yfh1Δ 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 3′-end of the YFH1 coding sequence, the 9E10 c-myc epitope coding sequence, a stop codon, 22 base pairs of the YFH1 3′-flanking DNA, and aBamHI site. This polymerase chain reaction product was substituted for the 3′-region of the YFH1 gene by digestion with AccI and BamHI, yielding aYFH1-myc fusion construct. A centromericTRP1-based YCplac22-YFH1-myc plasmid was then used to transform the yfh1Δ[YFH1] strain (16Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholar) and replace the URA3-based YCp50-YFH1plasmid, which was eliminated by counterselection with 5-fluoroorotic acid, yielding the yfh1Δ[YFH1-myc] strain.Table IS. cerevisiae strainsStrainGenotypeReferenceYPH501MATa/αura3–52/ura3–52 lys2–801 amber/lys2–801 amberade2–101 ochre/ade2–101 ochre trp1-Δ63/trp1-Δ63 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ115Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google ScholarY6041 (wild type)MATa ura3–52 lys2–801 amberade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ115Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholaroct1ΔMATα ura3–52 lys2–801 amberade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ1 oct1Δ∷LEU215Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholaryfh1ΔMATαura3–52 lys2–801 amber ade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ1 yfh1Δ∷HIS316Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholaryfh1Δ[YFH1]MATα ura3–52 lys2–801 amber ade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ1 yfh1Δ∷HIS3 + YC-YFH116Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholaryfh1Δ[YFH1-myc]MATa ura3–52 lys2–801 amber ade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ1 yfh1Δ∷HIS3 + YC-YFH1-mycThis studyIsogenic ρ0MATa ura3–52 lys2–801 amber ade2–101 ochre trp1-Δ63 his3-Δ200 leu2-Δ1 [rho 0]16Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholar Open table in a new tab Theyfh1Δ[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 anA 600 of ∼2, spheroplasts were prepared and homogenized, and the nuclear (1,000 × g pellet), heavy (3,000 × g pellet), and light (17,000 ×g pellet) mitochondrial fractions were separated by differential centrifugation. The light mitochondrial fraction was resuspended at 2 mg protein/ml in either isotonic (0.6 mmannitol, 20 mm HEPES-KOH, pH 7.4, 1 mg/ml bovine serum albumin) or hypotonic (20 mm HEPES-KOH, pH 7.4, 1 mg/ml bovine serum albumin) buffer and incubated for 25 min at 4 °C with gentle vortexing for 30 s every 5 min, essentially as described (17Rowley N. Prip-Buus C. Westermann B. Brown C. Schwarz E. Barrell B. Neupert W. Cell. 1994; 77: 249-259Abstract Full Text PDF PubMed Scopus (200) Google Scholar). When indicated, this treatment was carried out in the presence of 100 μg/ml proteinase K, with or without 1% Triton X-100. Proteinase K treatment was stopped by addition of 100 mmphenylmethylsulfonyl fluoride. Mitochondria were then resuspended in 20 mm HEPES-KOH, pH 7.4, 100 mm KCl and subjected to five cycles of freezing and thawing. Finally, the disrupted organelles were separated into soluble (matrix) and insoluble (membrane) fractions by centrifugation at 165,000 × g. Fractions were precipitated with 10% trichloroacetic acid (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar), protein concentration was determined by ultraviolet absorption (18Layne E. Methods Enzymol. 1957; 3: 447-454Crossref Scopus (2733) Google Scholar), and aliquots were analyzed by SDS/PAGE, Western blotting, and chemiluminescence. Yfh1p was detected using a monoclonal antibody against the 9E10 c-myc epitope or a polyclonal antibody (16Branda S.S. Yang Z. Chew A. Isaya G. Hum. Mol. Gen. 1999; 8: 1099-1110Crossref PubMed Scopus (55) Google Scholar); frataxin was detected using a polyclonal antibody against a GST-human frataxin fusion protein. 2P. Cavadini and F. Taroni, manuscript in preparation. Antisera against mitochondrial Hsp60 and cytochrome b 2 (Cytb 2) were gifts from other investigators. [35S]Methionine-labeled precursors were synthesized in vitro by coupled transcription-translation (Promega). Previously described procedures were used for isolation of yeast (19Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar) and rat liver (20Kalousek F. Isaya G. Rosenberg L.E. EMBO J. 1992; 11: 2803-2809Crossref PubMed Scopus (87) Google Scholar) mitochondria. Translation mixture (6 μl) containing 35S-labeled precursor was incubated with mitochondria (total protein, 80 μg) in import buffer (0.6m mannitol, 20 mm HEPES-KOH, pH 7.4, 1 mm ATP, 1 mm MgCl2, 40 mm KCl, 5 mm methionine, 3 mg/ml bovine serum albumin, 20 mm phosphocreatine, and 200 μg/ml phosphocreatine kinase) for 20 min at 27 °C (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar). Upon import, reactions were either separated into mitochondrial pellet and post-mitochondrial supernatant by centrifugation at 14,000 ×g for 5 min at 4 °C or first treated with proteinase K (250 μg/ml for 30 min at 0 °C) or trypsin (400 μg/ml for 5 min at 4 °C) and then separated into pellet and supernatant in the presence of protease inhibitors. To dissipate the inner membrane potential, mitochondria were incubated with 30 μmcarbonyl cyanide m-chlorophenyl-hydrazone for 5 min at 0 °C prior to import. Crude matrix fractions were prepared by sonication of mitochondria followed by centrifugation at 165,000 × g for 30 min (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar). Recombinant yeast MPP was prepared essentially as described by Geli (21Geli V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6247-6251Crossref PubMed Scopus (45) Google Scholar); as determined by SDS/PAGE and Coomassie blue staining, the final enzyme preparation contained only αMPP and βMPP subunits (>99% purity). 3J. Adamec, unpublished results. One unit of recombinant MPP was arbitrarily defined as the amount of enzyme that converts 95% of the yeast F1-ATPase subunit β precursor (pF1β) contained in 5 μl of translation mixture to the mature form in 5 min at 27 °C in a total reaction volume of 50 μl of 10 mm HEPES-KOH (pH 7.4), 1 mmdithiothreitol, 1 mm MnCl2 (HDM buffer). Import and processing reactions were directly analyzed by SDS/PAGE and fluorography. For analysis of Yfh1p and human frataxin processing, we used T = 12.5% separating gels (total length, 12.5 cm) overlaid with T = 4% stacking gels (Tdenotes the total concentration of acrylamide and bisacrylamide), from a stock solution of 40:1.7 acrylamide:bisacrylamide; electrophoresis was started at 180 V, shifted to 240 V after the samples had completely entered the separating gel, and continued for an additional 75 min (pYfh1p) or 30 min (frataxin) after the samples had reached the bottom of the separating gel. To analyze mitochondrial import and processing of Yfh1p, theYFH1 coding sequence was cloned into an in vitroexpression vector, and radiolabeled pYfh1p was synthesized by coupled transcription-translation. SDS/PAGE analysis of the translation mixture revealed a major band with an apparent molecular mass of ∼28 kDa, much larger than the predicted size of pYfh1p (∼19.5 kDa) (Fig.1 A, lane 1;lanes 8 and MW show the mobility of pYfh1p relative to those of standard proteins). A difference of almost 10 kDa between the predicted molecular mass and the electrophoretic mobility of pYfh1p was reported previously (12Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), and similar discrepancies were noted for human (10Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (616) Google Scholar) and mouse (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar) frataxin as well. Such differences are observed regardless of whether Yfh1p and frataxin are produced in intact cells or in vitro translation assays (Ref. 13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar and this study), and therefore it seems unlikely that they result from post-translational modifications. A more likely explanation is that the extremely hydrophilic nature of frataxin causes it to bind less SDS as compared with standard proteins of the same mass and that this results in lower electrophoretic mobility. In agreement with this interpretation, we show in this study that N-terminally deleted variants of Yfh1p migrate in SDS/PAGE at rates slower than predicted from their molecular masses but proportional to the number of deleted amino acids (see below). Incubation of radiolabeled pYfh1p with isolated yeast mitochondria yielded two major processing products with apparent molecular masses of ∼27 kDa and ∼21 kDa (designated intermediate (i), and mature (m), respectively) (Fig. 1 A, lane 2; lanes 8 and MW show the mobilities of iYfh1p and mYfh1p relative to those of standard proteins). Both iYfh1p and mYfh1p were associated with the mitochondrial pellet (lane 3) and were protected from externally added proteinase K (lane 5) but were degraded when protease treatment was performed in the presence of Triton X-100 (not shown). When the inner membrane potential was dissipated by addition of carbonyl cyanidem-chlorophenyl-hydrazone prior to import, the precursor was still associated with the mitochondrial pellet (lane 7) but was not protected from proteinase K (not shown), nor were the two processing products formed (lane 7). Thus, pYfh1p is specifically imported by isolated yeast mitochondria, and its translocation to a protease-protected compartment is associated with two proteolytic events. This pattern of processing sets pYfh1p apart from the vast majority of mitochondrial protein precursors, which are cleaved to the mature form in a single step by MPP (14Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (978) Google Scholar). On the other hand, two-step processing has been reported for precursors targeted to the intermembrane space, which are cleaved sequentially by MPP and the inner membrane peptidase (IMP) (22Schneider A. Behrens M. Scherer P. Pratje E. Michaelis G. Schatz G. EMBO J. 1991; 10: 247-254Crossref PubMed Scopus (136) Google Scholar), as well as for a subset of precursors targeted to the matrix or the inner membrane, which are processed by MPP and the mitochondrial intermediate peptidase (MIP; EC 3.4.24.59) (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar, 20Kalousek F. Isaya G. Rosenberg L.E. EMBO J. 1992; 11: 2803-2809Crossref PubMed Scopus (87) Google Scholar). In light of this, and considering that MPP was shown to catalyze conversion of the mouse frataxin precursor to intermediate form (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar), we investigated whether IMP or MIP might be involved in mYfh1p production. Because the proteins cleaved by these peptidases are targeted to specific mitochondrial compartments, we sought to define the intramitochondrial localization of mYfh1p. Polyclonal antibodies against Yfh1p were not available at the time of these experiments, and therefore a sequence encoding the c-myc epitope (9E10) was fused to the YFH1 coding sequence immediately upstream of the stop codon. A centromeric YC-YFH1-myc plasmid was then substituted for the YC-YFH1 plasmid in strainyfh1Δ[YFH1] (TableI for strain genotypes), yieldingyfh1Δ[YFH1-myc] derivatives that grew as well as the parental strain under a variety of conditions (not shown), indicating that the c-myc epitope does not affect Yfh1p function. Radiolabeled pYfh1p-myc was efficiently imported by isolated mitochondria and was cleaved to two products slightly larger than the iYfh1p and mYfh1p products generated upon import of untagged precursor (Fig. 1 B, lanes 9and 10), demonstrating that the c-myc epitope does not affect N-terminal processing of pYfh1p. Furthermore, it was previously reported that Yfh1p fused to five copies of the c-myc epitope localizes to mitochondria, as determined by immunofluorescence staining (3Wilson R.B. Roof D.M. Nat. Genet. 1997; 16: 352-357Crossref PubMed Scopus (326) Google Scholar). These data clearly indicate that Yfh1p-myc is fully functional, and consequently its intramitochondrial localization must reflect that of the native protein. We therefore isolated mitochondria from theyfh1Δ[YC-YFH1-myc] strain and analyzed by Western blotting the Yfh1p-myc distribution in mitochondrial subfractions. In intact mitochondria we detected a single protein band (Fig. 1 C, lane 11) that migrated identically to the mYfh1p-myc product formed upon import of radiolabeled pYfh1p-myc into isolated mitochondria (Fig.1 B, lane 10). Although the precursor form was not detected in vivo, overexposed blots did reveal low levels of iYfh1p-myc (not shown). The mYfh1p-myc product was associated with intact mitochondria (Fig. 1 C, lane 11) as well as mitochondria subjected to hypotonic shock (to disrupt the outer mitochondrial membrane) (lane 13); furthermore, mYfh1p-myc was protected when mitochondria were subjected to proteinase K treatment (lane 12) or both hypotonic shock and proteinase K treatment (lane 14). In contrast, mYfh1p-myc was fully degraded when proteinase K was added to mitochondria in the presence of Triton X-100 (lane 15), indicating that protection of mYfh1p-myc from protease treatment requires an intact inner mitochondrial membrane. The mYfh1p-myc product was recovered in the soluble fraction derived from mitochondria that were subjected to hypotonic shock and then repeated cycles of freezing and thawing (to gently disrupt the inner membrane) (lane 16). This was also the case when the soluble fraction was derived from mitochondria that were subjected to both hypotonic shock and proteinase K treatment (to degrade any proteins external to the inner membrane) prior to freezing and thawing (lane 18). This fractionation pattern was similar to that of Hsp60, a soluble matrix protein (23Cheng M.Y. Hartl F.U. Horwich A.L. Nature. 1990; 348: 455-458Crossref PubMed Scopus (149) Google Scholar), but different from that of Cytb 2, a soluble intermembrane space protein (24Glick B.S. Brandt A. Cunningham K. Muller S. Hallberg R.L. Schatz G. Cell. 1992; 69: 809-822Abstract Full Text PDF PubMed Scopus (301) Google Scholar) that was fully accessible to proteinase K after hypotonic shock (lanes 14 and 18). Furthermore, mYfh1p-myc partitioned differently from the Rieske iron-sulfur protein, an inner membrane protein (25Graham L.A. Brandt U. Sargent J.S. Trumpower B.L. J. Bioenerg. Biomembr. 1993; 25: 245-257Crossref PubMed Scopus (58) Google Scholar) that was detected primarily in the membrane fractions (not shown). Thus, mYfh1p-myc behaved like a soluble mitochondrial matrix protein, and this result was confirmed for endogenous mYfh1p using a polyclonal antibody (not shown). Although localization to the matrix is consistent with the hydrophilic nature of Yfh1p (2Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (347) Google Scholar), Campuzano et al. (10Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (616) Google Scholar) showed by immunoelectronmicroscopy that human frataxin localizes at or near the inner mitochondrial membrane. Therefore, we cannot exclude the possibility that mYfh1p is loosely bound to the inner mitochondrial membrane in intact mitochondria and that this interaction is disrupted when mitochondria are fractionated. In any case, our results clearly indicate that mYfh1p is not localized to the intermembrane space and therefore exclude the possibility that IMP is involved in the maturation of Yfh1p. On the other hand, the fact that mYfh1p localizes to the matrix is consistent with the possibility that pYfh1p is processed in two sequential steps by two matrix-localized peptidases, MPP and MIP. Precursors cleaved by these two peptidases are characterized by a three-amino acid motif, RX↓(F/L/I)XX(S/T/G)XXXX↓, at the C terminus of their leader peptide (26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27Hendrick J.P. Hodges P.E. Rosenberg L.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4056-4060Crossref PubMed Scopus (264) Google Scholar, 28Gavel Y. Von Heijne G. Protein Eng. 1990; 4: 33-37Crossref PubMed Scopus (275) Google Scholar). MPP initially cleaves these precursors two peptide bonds C-terminal to the Arg residue in the motif, yielding a processing intermediate with a typical N-terminal octapeptide, which is then specifically removed by MIP to yield the mature protein (29Isaya G. Kalousek F. Fenton W.A. Rosenberg L.E. J. Cell Biol. 1991; 113: 65-76Crossref PubMed Scopus (104) Google Scholar). The N-terminal region of neither pYfh1p nor the frataxin precursor contains this motif, however, suggesting that MIP is not involved in their maturation. In fact, radiolabeled pYfh1p was imported and processed to the mature form by mitochondria isolated from a knock-out mutant (oct1Δ) lacking yeast MIP (OCT1, gene; YMIP, polypeptide) 4The open reading frame YKL134C, encoding the yeast mitochondrial intermediate peptidase, has recently been renamedOCT1 (YMIP, polypeptide). OCT1 was previously referred to as MIP1 (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar), but this name was first assigned to open reading frame YOR330C. (Fig.2 A, lane 2). In contrast, oct1Δ mitochondria did not cleave the intermediate form of the cytochrome c oxidase subunit IV (iCoxIV) (Fig. 2 B, lane 2), which is normally processed to the mature form by YMIP (15Isaya G. Miklos D. Rollins R.A. Mol. Cell. Biol. 1994; 14: 5603-5616Crossref PubMed Scopus (68) Google Scholar, 26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Interestingly,oct1Δ mitochondria produced less mature protein than did wild-type mitochondria (Fig. 2 A, compare lanes 1and 2), and in this respect behaved identically to mitochondria isolated from an isogenic ρ0 strain (lane 3). Similarly, pCoxIV was inefficiently processed byoct1Δ and ρ0 mitochondria (Fig.2 B, lanes 2 and 3), confirming that reduced production of mYfh1p by oct1Δ mitochondria is not indicative of a specific involvement of YMIP in pYfh1p processing. Rather, this effect probably results from loss of mitochondrial DNA inoct1Δ (26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), a condition that is known to affect the efficiency of in vitro import assays (30Yaffe M.P. Methods Enzymol. 1991; 194: 627-643Crossref PubMed Scopus (150) Google Scholar). In agreement with these in vitro results, endogenous mYfh1p was detected by Western analysis of mitochondria isolated from oct1Δ yeast (Fig. 2 C, lane 5), further demonstrating that YMIP is not directly involved in the maturation of Yfh1p. Having excluded direct participation of IMP or MIP in Yfh1p processing, we tested the possibility that MPP might be solely responsible for production of both iYfh1p and mYfh1p. To determine whether typical MPP cleavage sites (26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27Hendrick J.P. Hodges P.E. Rosenberg L.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4056-4060Crossref PubMed Scopus (264) Google Scholar, 28Gavel Y. Von Heijne G. Protein Eng. 1990; 4: 33-37Crossref PubMed Scopus (275) Google Scholar) are used in the processing of pYfh1p, we synthesized a series of N-terminally truncated versions of pYfh1p and used them as standards to map the N termini of iYfh1p and mYfh1p. In our SDS/PAGE system, iYfh1p ran slightly faster than a product translated from residue 21 of pYfh1p (designated M-iYfh1p) but slower than a product translated from residue 25 (Fig.3). Thus, the first cleavage site must lie between residues 21 and 25, and indeed residues 19–22 (RYM ↓I) match the consensus sequence RX(X/Y)↓(X/A/S), which is found at many MPP cleavage sites (26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Gavel Y. Von Heijne G. Protein Eng. 1990; 4: 33-37Crossref PubMed Scopus (275) Google Scholar) (Fig. 3). Similarly, mYfh1p ran between products translated from residues 52 and 56, and residues 50–53 (RFV↓E) also match the RX(X/Y) ↓(X/A/S) consensus sequence (Fig. 3). Moreover, the amino acids C-terminal to this putative MPP cleavage site include two serines and one threonine, which is consistent with observations that the mature N termini of mitochondrial proteins frequently contain small hydroxylated residues (26Branda S.S. Isaya G. J. Biol. Chem. 1995; 270: 27366-27373Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Gavel Y. Von Heijne G. Protein Eng. 1990; 4: 33-37Crossref PubMed Scopus (275) Google Scholar). To confirm that pYfh1p is indeed processed in two steps by MPP, radiolabeled pYfh1p was incubated with recombinant yeast MPP, which was reconstituted from bacterially expressed and purified subunits using a procedure similar to that described previously by Geli (21Geli V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6247-6251Crossref PubMed Scopus (45) Google Scholar). After 10 min of incubation (Fig. 4 A,lane 1), most of the input pYfh1p was no longer detected, whereas iYfh1p was accumulated along with smaller amounts of mYfh1p; incubation for an additional 10 min (lane 2) resulted in increased production of mYfh1p with concomitant disappearance of iYfh1p. Given that most of the precursor was converted to iYfh1p during the first 10 min of incubation (lane 1), we conclude that the mYfh1p accumulated in the subsequent 10 min (lane 2) was produced by cleavage of iYfh1p. The fact that disappearance of the precursor band was not associated with a proportional increase in the intensity of the iYfh1p and mYfh1p bands (lane 1) can be explained by the loss of three of the four radiolabeled methionine residues present in the precursor sequence (codons 1, 16, and 21) upon processing to intermediate form (Fig. 3). On the other hand, iYfh1p and mYfh1p are each predicted to contain a single methionine residue (codon 109), and disappearance of the accumulated iYfh1p (lane 1) coincided with formation of an equal amount of mYfh1p (lane 2). Apparently identical products were generated whether pYfh1p was incubated with recombinant MPP (lanes 1–3), total mitochondrial matrix (lane 4), or isolated mitochondria (lane 5). It is important to note, however, that whereas mYfh1p was efficiently produced in isolated mitochondria (lane 5), only trace amounts of mYfh1p were produced by matrix (lane 4). Similarly, although 10-fold lower concentrations of MPP were sufficient for processing of pYfh1p to iYfh1p, higher enzyme levels and longer incubation times were required for conversion of iYfh1p to mYfh1p (not shown). The possibility that under these experimental conditions recombinant MPP might have cleaved iYfh1p nonspecifically seems unlikely for two reasons: first, under very similar conditions pCoxIV was processed to intermediate form only (Fig.4 B, lane 6); and second, pF1β was processed to the mature form after only 5 min of incubation with MPP (Fig. 4 C, lane 9), but no further proteolysis occurred during an additional 25 min of incubation at 27 °C (lanes 10–13). Thus, a more likely explanation is that whereas MPP per se is sufficient to catalyze conversion of iYfh1p to mYfh1p, additional factors such as mitochondrial membrane integrity may affect the efficiency of this reaction. In fact, Knightet al. (12Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) showed previously that Ssq1p, a mitochondrial Hsp70 homologue, is required for formation of mYfh1p in vivo, suggesting that factors influencing the conformation of iYfh1p may affect the rate of its conversion to mature form. To further confirm that mYfh1p is produced from iYfh1p, we analyzed processing of the N-terminally truncated product translated from residue 21 of pYfh1p (M-iYfh1p), which is predicted to be one amino acid longer than iYfh1p (Fig. 3). We found that M-iYfh1p was processed to the mature form very efficiently by recombinant MPP (Fig.4 D, lane 14), and less efficiently by matrix (lane 16), a processing pattern similar to that observed for iYfh1p (Fig. 4 A, lanes 3 and 4). The mature size product generated by cleavage of M-iYfh1p in these reactions (lanes 14 and 16) was indistinguishable from the mYfh1p produced upon import of pYfh1p into isolated yeast mitochondria (lane 15). This result provides further support to the conclusion that MPP first cleaves the Yfh1p precursor to the intermediate form and then converts this product to the mature form. Although two-step processing by MPP has been described for at least one other mitochondrial protein precursor (31Hawlitschek G. Schneider H. Schmidt B. Tropschug M. Hartl F.U. Neupert W. Cell. 1988; 53: 785-806Abstract Full Text PDF Scopus (1) Google Scholar), our observations do not agree with those of a previous study in which recombinant rat MPP appeared to process the precursor of mouse frataxin to the intermediate but not the mature form (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar). One possible explanation for this discrepancy might be that rather than a purified enzyme, the previous study used crude extracts of bacterial cells that co-expressed both MPP subunits and perhaps a factor in these extracts inhibited the second cleavage. To test this possibility, we analyzed the processing of [35S]methionine-labeled human frataxin precursor, the sequence of which is almost identical to that of mouse frataxin (4Koutnikova H. Campuzano V. Foury F. Dollé P. Cazzalini O. Koenig M. Nat. Genet. 1997; 16: 345-351Crossref PubMed Scopus (430) Google Scholar). Because the human frataxin sequence does not contain any methionine residues C-terminal to codon 76, we used a construct containing an in-frame C-terminal tag of 10 amino acids that includes a methionine residue (32Cavadini P. DiBlasi C. Princivalle A. Baratta S. DiDonato S. Battaglia G. Mora M. Taroni F. Am. J. Hum. Genet. 1997; 61 (abstr.): 42Google Scholar). Upon incubation with recombinant yeast MPP, most of the input precursor was converted to the intermediate form (Fig.5, lane 1); we also detected trace amounts of a smaller product with a mobility similar to that reported for mature frataxin (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar) (lane 1). Addition of a fresh aliquot of MPP resulted in modestly increased conversion of the accumulated intermediate to the putative mature form (lane 2). This result was reproduced in three independent experiments, and a very similar pattern of processing was observed when the frataxin precursor was incubated with rat liver mitochondria (lane 3) or bacterially expressed recombinant rat MPP (not shown). To exclude the possibility that the C-terminal tag might interfere with cleavage of intermediate frataxin to the mature form, the wild-type frataxin precursor (i.e. lacking the C-terminal tag) was incubated with yeast MPP as described above, and processing reactions were analyzed by Western blotting. As was the case for the tagged precursor, we detected a major processing product corresponding to the intermediate form of frataxin and only trace amounts of the putative mature form (Fig. 5 B, lane 5). The latter product migrated identically to mature frataxin, as detected in a variety of human tissue extracts (lane 6 and not shown). Thus, under our experimental conditions, MPP did efficiently cleave the frataxin precursor to the intermediate form but could only partially process this intermediate to mature size protein. Given that the intermediate form of frataxin was processed very inefficiently even upon import into rat mitochondria (Fig. 5 A, lane 3), it seems that similar to Yfh1p, frataxin is processed in two sequential steps by MPP and that species- and/or tissue-specific factors are involved in the second cleavage. A number of recent studies indicate that Yfh1p and frataxin play conserved roles in mitochondrial iron homeostasis and free radical toxicity (1Babcock M. De Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar, 2Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (347) Google Scholar, 3Wilson R.B. Roof D.M. Nat. Genet. 1997; 16: 352-357Crossref PubMed Scopus (326) Google Scholar, 4Koutnikova H. Campuzano V. Foury F. Dollé P. Cazzalini O. Koenig M. Nat. Genet. 1997; 16: 345-351Crossref PubMed Scopus (430) Google Scholar, 5Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 6Lamarche J.B. Côté M. Lemieux B. J. Can. Sci. Neurol. 1980; 7: 389-396Crossref PubMed Scopus (173) Google Scholar, 7Rötig A. De Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genet. 1997; 17: 215-217Crossref PubMed Scopus (880) Google Scholar, 8Wong A. Yang J. Cavadini P. Gellera C. Lonnerdal B. Taroni F. Cortopassi G. Hum. Mol. Genet. 1999; 8: 425-430Crossref PubMed Scopus (326) Google Scholar), supporting a model in which frataxin deficiency results in oxidative damage, which in turn leads to the degenerative lesions of FRDA (11Pandolfo M. Neuromusc. Dis. 1998; 8: 409-415Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Moreover, the clinical variability observed in FRDA patients suggests that additional pathogenetic factors, such as mitochondrial proteins that interact with frataxin, may influence the phenotypic expression of frataxin deficiency (11Pandolfo M. Neuromusc. Dis. 1998; 8: 409-415Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). MPP was previously identified as the peptidase responsible for one of two cleavages required for the biogenesis of mouse frataxin (13Koutnikova H. Campuzano V. Koenig M. Hum. Mol. Gen. 1998; 7: 1485-1489Crossref PubMed Scopus (102) Google Scholar), and we have demonstrated that MPP is solely responsible for two-step processing of yeast and human frataxin. Knight et al. (12Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) also identified Ssq1p, a mitochondrial Hsp70 homologue, as an additional factor required for cleavage of iYfh1p to mature form. Thus, it is tempting to speculate that this pattern of processing has a regulatory function and that genetic or environmental factors that influence the affinity of MPP for the frataxin intermediate might play a role in iron homeostasis and the clinical manifestations of FRDA. We thank A. L. Horwich and G. Schatz for antibodies.
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