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• W1988367050 abstract "Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the β-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of β-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space. Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the β-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of β-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space. Mitochondria contain ∼800–1500 different proteins (1Taylor S.W. Fahy E. Zhang B. Glenn G.M. Warnock D.E. Wiley S. Murphy A.N. Gaucher S.P. Capaldi R.A. Gibson B.W. Ghosh S.S. Nature Biotechnol. 2003; 21: 281-286Google Scholar, 2Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schönfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Google Scholar, 3Westermann B. Neupert W. Nature Biotechnol. 2003; 21: 239-240Google Scholar, 4Mootha V.K. Bunkenborg J. Olsen J.V. Hjerrild M. Wisniewski J.R. Stahl E. Bolouri M.S. Ray H.N. Sihag S. Kamal M. Patterson N. Lander E.S. Mann M. Cell. 2003; 115: 629-640Google Scholar). 99% of these proteins are encoded in the nucleus and synthesized as precursors on cytosolic ribosomes (5Koehler C.M. Merchant S. Schatz G. Trends Biochem. Sci. 1999; 24: 428-432Google Scholar, 6Bauer M.F. Hofmann S. Neupert W. Brunner M. Trends Cell Biol. 2000; 10: 25-31Google Scholar, 7Matouschek A. Pfanner N. Voos W. EMBO Rep. 2000; 1: 404-410Google Scholar, 8Jensen R.E. Johnson A.E. Nat. Struct. Biol. 2001; 8: 1008-1010Google Scholar, 9Pfanner N. Chacinska A. Biochim. Biophys. Acta. 2002; 1592: 15-24Google Scholar). The precursor proteins are recognized by receptors of the mitochondrial outer membrane and are translocated by the general import pore (GIP) 1The abbreviations used are: GIP, general import pore; TOM, translocase of outer mitochondrial membrane; TIM, translocase of inner mitochondrial membrane; DHFR, dihydrofolate reductase; SAM, sorting and assembly machinery. across the membrane. The receptors and the GIP assemble to form a high molecular weight complex, termed the translocase of the outer mitochondrial membrane (TOM complex) (10Yano M. Hoogenraad N. Terada K. Mori M. Mol. Cell. Biol. 2000; 20: 7205-7213Google Scholar, 11Gabriel K. Buchanan S.K. Lithgow T. Trends Biochem. Sci. 2001; 26: 36-40Google Scholar, 12Endo T. Kohda D. Biochim. Biophys. Acta. 2002; 1592: 3-14Google Scholar, 13Pfanner N. Wiedemann N. Curr. Opin. Cell Biol. 2002; 14: 400-411Google Scholar, 14Rapaport D. Trends Biochem. Sci. 2002; 27: 191-197Google Scholar). The three receptors, Tom20, Tom22, and Tom70, show a distinct yet overlapping specificity for different classes of mitochondrial precursor proteins. With the help of the small Tom protein, Tom5, the precursors are transferred to the core of the TOM complex, the GIP formed by the essential protein Tom40 (15Dietmeier K. Hönlinger A. Bömer U. Dekker P.J.T. Eckerskorn C. Lottspeich F. Kübrich M. Pfanner N. Nature. 1997; 388: 195-200Google Scholar, 16Hill K. Model K. Ryan M.T. Dietmeier K. Martin F. Wagner R. Pfanner N. Nature. 1998; 395: 516-521Google Scholar). Two additional small Tom proteins, Tom6 and Tom7, are involved in the stability and assembly of the TOM complex. Tom40, the three small Tom proteins, and the receptors Tom22 and Tom20 are associated in a complex of about 450 kDa, whereas the receptor Tom70 is only loosely associated with the other Tom proteins (17Dekker P.J.T. Ryan M.T. Brix J. Müller H. Hönlinger A. Pfanner N. Mol. Cell. Biol. 1998; 18: 6515-6524Google Scholar, 18Künkele K.P. Heins S. Dembowski M. Nargang F.E. Benz R. Thieffry M. Walz J. Lill R. Nussberger S. Neupert W. Cell. 1998; 93: 1009-1019Google Scholar, 19van Wilpe S. Ryan M.T. Hill K. Maarse A.C. Meisinger C. Brix J. Dekker P.J.T. Moczko M. Wagner R. Meijer M. Guiard B. Hönlinger A. Pfanner N. Nature. 1999; 401: 485-489Google Scholar, 20Stan T. Ahting U. Dembowski M. Künkele K.P. Nussberger S. Neupert W. Rapaport D. EMBO J. 2000; 19: 4895-4902Google Scholar, 21Meisinger C. Ryan M.T. Hill K. Model K. Lim J.H. Sickmann A. Müller H. Meyer H.E. Wagner R. Pfanner N. Mol. Cell. Biol. 2001; 21: 2337-2348Google Scholar, 22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar). After passing the TOM complex, the precursor proteins are transferred to the other mitochondrial compartments, the intermembrane space, inner membrane, and matrix. Two main classes of precursor proteins can be distinguished (5Koehler C.M. Merchant S. Schatz G. Trends Biochem. Sci. 1999; 24: 428-432Google Scholar, 6Bauer M.F. Hofmann S. Neupert W. Brunner M. Trends Cell Biol. 2000; 10: 25-31Google Scholar, 7Matouschek A. Pfanner N. Voos W. EMBO Rep. 2000; 1: 404-410Google Scholar, 8Jensen R.E. Johnson A.E. Nat. Struct. Biol. 2001; 8: 1008-1010Google Scholar, 13Pfanner N. Wiedemann N. Curr. Opin. Cell Biol. 2002; 14: 400-411Google Scholar). Preproteins with amino-terminal cleavable targeting sequences (presequences) are transferred from the TOM complex to the presequence translocase (TIM23 complex) of the inner membrane and its associated protein import motor, PAM. Non-cleavable precursor proteins with multiple internal targeting signals, like the hydrophobic metabolite carriers of the inner membrane, are transferred from the TOM complex to small Tim proteins of the intermembrane space that guide them through this aqueous compartment. The hydrophobic proteins are then delivered to the protein insertion machinery (carrier translocase, TIM22 complex) of the inner membrane (5Koehler C.M. Merchant S. Schatz G. Trends Biochem. Sci. 1999; 24: 428-432Google Scholar, 6Bauer M.F. Hofmann S. Neupert W. Brunner M. Trends Cell Biol. 2000; 10: 25-31Google Scholar, 23Jensen R.E. Dunn C.D. Biochim. Biophys. Acta. 2002; 1592: 25-34Google Scholar, 24Rehling P. Pfanner N. Meisinger C. J. Mol. Biol. 2003; 326: 639-657Google Scholar). Two soluble complexes of small Tim proteins are known in the intermembrane space, the essential Tim9-Tim10 complex and the Tim8-Tim13 complex (25Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Google Scholar, 26Sirrenberg C. Endres M. Fölsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Google Scholar, 27Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Google Scholar, 28Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Google Scholar, 29Koehler C.M. Leuenberger D. Merchant S. Renold A. Junne T. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2141-2146Google Scholar, 30Kurz M. Martin H. Rassow J. Pfanner N. Ryan M.T. Mol. Biol. Cell. 1999; 10: 2461-2474Google Scholar, 31Luciano P. Vial S. Vergnolle M.A.S. Dyall S.D. Robinson D.R. Tokatlidis K. EMBO J. 2001; 20: 4099-4106Google Scholar, 32Curran S.P. Leuenberger D. Oppliger W. Koehler C.M. EMBO J. 2002; 21: 942-953Google Scholar, 33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar). The Tim9-Tim10 complex is involved in transfer of many hydrophobic proteins through the intermembrane space, whereas the Tim8-Tim13 complex plays a special role for a few precursor proteins, like the precursor of the inner membrane protein Tim23 (34Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Google Scholar, 35Davis A.J. Sepuri N.B. Holder J. Johnson A.E. Jensen R.E. J. Cell Biol. 2000; 150: 1271-1282Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar, 37Rothbauer U. Hofmann S. Mühlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Google Scholar, 38Curran S.P. Leuenberger D. Schmidt E. Koehler C.M. J. Cell Biol. 2002; 158: 1017-1027Google Scholar). All mitochondrial outer membrane proteins are encoded by nuclear genes, synthesized in the cytosol and imported by means of the TOM complex. Thus the precursors of Tom proteins require the pre-existing mature TOM complex for entry into mitochondria (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 39Schneider H. Söllner T. Dietmeier K. Eckerskorn C. Lottspeich F. Trülzsch B. Neupert W. Pfanner N. Science. 1991; 254: 1659-1662Google Scholar, 40Keil P. Pfanner N. FEBS Lett. 1993; 321: 197-200Google Scholar, 41Keil P. Weinzierl A. Kiebler M. Dietmeier K. Söllner T. Pfanner N. J. Biol. Chem. 1993; 268: 19177-19180Google Scholar, 42Rapaport D. Neupert W. J. Cell Biol. 1999; 146: 321-331Google Scholar, 43Dembowski M. Künkele K.P. Nargang F.E. Neupert W. Rapaport D. J. Biol. Chem. 2001; 276: 17679-17685Google Scholar, 44Johnston A.J. Hoogenraad J. Dougan D.A. Truscott K.N. Yano M. Mori M. Hoogenraad N.J. Ryan M.T. J. Biol. Chem. 2002; 277: 42197-42204Google Scholar, 45Taylor R.D. McHale B.J. Nargang F.E. J. Biol. Chem. 2003; 278: 765-775Google Scholar). The precursor of the GIP-forming β-barrel protein Tom40 is imported and assembled into the TOM complex by means of a multi-step pathway. The Tom40 precursor is recognized by Tom receptors (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 41Keil P. Weinzierl A. Kiebler M. Dietmeier K. Söllner T. Pfanner N. J. Biol. Chem. 1993; 268: 19177-19180Google Scholar, 42Rapaport D. Neupert W. J. Cell Biol. 1999; 146: 321-331Google Scholar) and transported by the GIP, i.e. pre-existing mature Tom40 (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). Subsequently, the precursor of Tom40 leaves the TOM complex and undergoes two maturation steps, termed assembly intermediate I and assembly intermediate II. The intermediates can be monitored by blue native electrophoresis of lysed mitochondria as 250- and 100-kDa complexes, respectively (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar). The assembly intermediate I is formed by the sorting and assembly machinery (SAM complex) (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 47Mihara K. Nature. 2003; 424: 505-506Google Scholar). The SAM complex contains the subunits Mas37 and Sam50 (Tob55/Omp85) (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 48Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Google Scholar, 49Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Google Scholar, 50Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Google Scholar). Subsequently, Tom40 stably associates with Tom5, forming the assembly intermediate II (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). Then Tom6, Tom7, Tom22, and finally Tom20 associate to form the mature TOM complex (21Meisinger C. Ryan M.T. Hill K. Model K. Lim J.H. Sickmann A. Müller H. Meyer H.E. Wagner R. Pfanner N. Mol. Cell. Biol. 2001; 21: 2337-2348Google Scholar, 22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 39Schneider H. Söllner T. Dietmeier K. Eckerskorn C. Lottspeich F. Trülzsch B. Neupert W. Pfanner N. Science. 1991; 254: 1659-1662Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). Here we report a surprising connection between two mitochondrial protein biogenesis pathways that have been assumed to represent separate entities so far, the SAM assembly pathway for Tom40 and the system of small Tim proteins that transfer the precursors of hydrophobic inner membrane proteins across the intermembrane space. The results presented here suggest that the intermembrane space is involved in the insertion of Tom40 into the outer membrane. Yeast Strains, Media, Mitochondrial Isolation, and Swelling—The Saccharomyces cerevisiae strains used in this study are the wild-type strain YPH499 (51Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Google Scholar) and the corresponding mutant strains tim8Δ tim13Δ (PRY34) and tim10–2 (GB102) (33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar). Yeast cells were grown on YPG medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 3% (v/v) glycerol) at 24 °C or 30 °C. Mitochondria were isolated by differential centrifugation as described (52Daum G. Böhni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Google Scholar, 53Hartl F.U. Ostermann J. Guiard B. Neupert W. Cell. 1987; 51: 1027-1037Google Scholar), and aliquots were stored at –80 °C in SEM buffer (250 mm sucrose, 1 mm EDTA, 10 mm MOPS-KOH, pH 7.2). For swelling of mitochondria, the pelleted mitochondria were resuspended in 1 vol of SEM buffer, and the suspension was diluted with 9 vol of swelling buffer (1 mm EDTA, 10 mm MOPS-KOH, pH 7.2) and incubated for 30 min on ice. Import of Preproteins—Radiolabeled precursor proteins were obtained by in vitro transcription from pGEM-4Z (or genomic PCR products containing an SP6 Promotor) using SP6 RNA polymerase (Stratagene) (54Ryan M.T. Voos W. Pfanner N. Methods Cell Biol. 2001; 65: 189-215Google Scholar). In vitro translation was performed with rabbit reticulocyte lysate in the presence of [35S]methionine/cysteine (Amersham Biosciences). Import assays were carried out with 25–50 μg of mitochondria (protein amount) in 100–200 μl of import buffer (3% (w/v) fatty acid-free bovine serum albumin, 250 mm sucrose, 80 mm KCl, 5 mm MgCl2, 10 mm MOPS-KOH, pH 7.2, 2–5 mm ATP, and, in the case of matrix precursor proteins, 2 mm NADH) with 2.5–10% (v/v) reticulocyte lysate. Import of precursor proteins was performed at 25 °C, except for the precursor of Tom20, where 10 °C was used (at 25 °C, import and assembly of Tom20 are too fast to be resolved in a kinetic manner). Where indicated, mitochondria were treated with 20–50 μg/ml proteinase K or 20 μg/ml trypsin for 15 min on ice. The proteases were inactivated by the addition of 1% (v/v) 200 mm phenylmethylsulfonyl fluoride/ethanol or 200 μg/ml soybean trypsin inhibitor, respectively. After a 5-min incubation on ice, the mitochondria were reisolated by centrifugation and washed with SEM buffer. Mitochondrial proteins were separated by glycine-SDS-PAGE, Tris-Tricine-SDS-PAGE, or blue native PAGE (17Dekker P.J.T. Ryan M.T. Brix J. Müller H. Hönlinger A. Pfanner N. Mol. Cell. Biol. 1998; 18: 6515-6524Google Scholar, 55Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Google Scholar). Miscellaneous—For affinity purification of anti-Tim9 antibodies, S. cerevisiae Tim9 was expressed in Escherichia coli strain BL21-Codon plus (DE3)-RIL (Stratagene) from the plasmid pET10N to produce an N-terminal His10-tagged protein. Cultures were grown at 37 °C for 4 h after induction of expression with 1 mm isopropyl-β-d-thiogalactopyranoside (IPTG). Tim9 was purified from isolated inclusion bodies under denaturing conditions via nickel-nitrilotriacetic acid-agarose chromatography essentially as described for Tim23 (56Truscott K.N. Kovermann P. Geissler A. Merlin A. Meijer M. Driessen A.J. Rassow J. Pfanner N. Wagner R. Nat. Struct. Biol. 2001; 8: 1074-1082Google Scholar), then dialysed against coupling buffer (30 mm Hepes-NaOH, pH 7.2, 2.5% (w/v) SDS) overnight at room temperature. Tim9 was coupled to CNBr-activated Sepharose 4B (Amersham Biosciences), as described by the manufacturer. To purify anti-Tim9 antibodies raised against an N-terminal region (peptide ALNSKEQQEFQKVVE), serum was diluted 1:1 with 1× TBS (10 mm Tris-HCl, pH 7.4, 0.9% (w/v) NaCl) then poured over the Tim9-Sepharose column. The column was washed extensively with 1× TBS prior to elution of specific antibodies with 100 mm glycine-HCl, pH 2.5. Samples were immediately neutralized with Tris buffer. Standard protocols were used for Western blotting and detection of immune complexes by enhanced chemiluminescence (Amersham Biosciences). Non-relevant gel lanes were excised digitally. Radiolabeled proteins were analyzed by digital autoradiography using the PhosphorImage technology (Amersham Biosciences). Opening of the Mitochondrial Intermembrane Space Blocks the Biogenesis Pathway of Tom40 but not that of Tom20 —The precursor of yeast Tom40 was synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine/cysteine. The precursor was incubated with isolated yeast mitochondria, and the assembly complexes were monitored by lysis of the mitochondria with digitonin and separated by blue native electrophoresis. In a time-dependent manner, the assembly intermediate I was formed, followed by the assembly intermediate II and the mature TOM complex, as described (Fig. 1A, lanes 1–3; Refs. 22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 48Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Google Scholar). To address a possible role of the intermembrane space in the biogenesis of Tom40, isolated mitochondria were subjected to swelling under conditions that led to an opening of the intermembrane space, whereas the inner membrane and matrix remained intact (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 57Geissler A. Chacinska A. Truscott K.N. Wiedemann N. Brandner K. Sickmann A. Meyer H.E. Meisinger C. Pfanner N. Rehling P. Cell. 2002; 111: 507-518Google Scholar). The swollen mitochondria (mitoplasts) were then incubated with the radiolabeled precursor of Tom40. The assembly pathway of Tom40 was dramatically affected by swelling because the formation of the assembly intermediate I was already blocked (Fig. 1A, lanes 4–6). Under these swelling conditions, neither the mature TOM complex nor the SAM complex were disturbed (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). The ruptured outer membrane remained attached to the mitoplasts, as shown by a Western blot analysis of endogenous Tom40 and Tom22, in comparison to Tim22 of the inner membrane (Fig. 1B). To analyze directly the intactness of the TOM complex, we imported the precursor of Tom20 that associates with the mature TOM complex. The assembly of Tom20 was not affected by the swelling of mitochondria (Fig. 1C). As an additional control, we show that the import of the matrix-targeted precursor of F1-ATPase subunit β was even moderately enhanced in mitoplasts (Fig. 1D, lanes 5–7; Ref. 58Kübrich M. Rassow J. Voos W. Pfanner N. Hönlinger A. J. Biol. Chem. 1998; 273: 16374-16381Google Scholar), compared with intact mitochondria (Fig. 1D, lanes 1–3). We conclude that opening of the intermembrane space blocks the assembly pathway of the Tom40 precursor, whereas other mitochondrial precusor proteins are still efficiently imported. Mutant Mitochondria of Small Tim Proteins Are Impaired in the Biogenesis Pathway of Tom40 —We used a yeast strain where both TIM8 and TIM13 were deleted (33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar, 59Frazier A.E. Chacinska A. Truscott K.N. Guiard B. Pfanner N. Rehling P. Mol. Cell. Biol. 2003; 23: 7818-7828Google Scholar). The assembly of Tom40 still took place, however, but with a reduced efficiency. In particular, the assembly intermediate I was reduced (Fig. 2A, lanes 7 and 8 versus lanes 2 and 3) and the formation of the mature TOM complex was delayed (Fig. 2A, lanes 9 and 10 versus lanes 4 and 5). To exclude indirect effects of the tim8Δ tim13Δ mitochondria, we analyzed the steady-state levels of various marker proteins. Except for the lack of the Tim8-Tim13 complex, all other proteins studied were present in roughly wild-type amounts, including the receptors Tom22, Tim9, and Tim23 of the inner membrane, and the matrix heat shock protein 70 (Fig. 2B). Blue native electrophoresis showed that the pre-existing TOM complex as well as the SAM complex were intact in tim8Δ tim13Δ mitochondria (Fig. 2C). Radiolabeled Tom20 assembled into the TOM complex of tim8Δ tim13Δ mitochondria with an efficiency close to that of wild-type mitochondria (Fig. 2D). Thus the delayed assembly of Tom40 cannot be attributed to a lack of TOM complex or SAM complex, supporting the conclusion that it was specifically due to the lack of the Tim8-Tim13 complex. Yeast cells lacking Tim8-Tim13 are viable (29Koehler C.M. Leuenberger D. Merchant S. Renold A. Junne T. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2141-2146Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar). As Tom40 is essential for cell viability (60Baker K.P. Schaniel A. Vestweber D. Schatz G. Nature. 1990; 348: 605-609Google Scholar), the Tim8-Tim13 complex can only play a supportive (accelerating) role in the assembly pathway of Tom40. Because the Tim9-Tim10 complex is essential for viability of yeast (25Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Google Scholar, 26Sirrenberg C. Endres M. Fölsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Google Scholar, 27Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Google Scholar, 28Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Google Scholar), we asked if this complex may also be required for the biogenesis of Tom40. We used the yeast mutant tim10–2 that carries point mutations in the TIM10 gene, causing a temperature-sensitive growth phenotype (33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar, 59Frazier A.E. Chacinska A. Truscott K.N. Guiard B. Pfanner N. Rehling P. Mol. Cell. Biol. 2003; 23: 7818-7828Google Scholar). When the cells were grown at the permissive temperature of 24 °C, the isolated mitochondria contained wild-type amounts of the marker proteins analyzed, including Tom22, Tim13, Tim23, and matrix heat shock protein 70. Only the amount of Tim9 was reduced by about half (Fig. 3A). Moreover, the TOM complex as well as the SAM complex were present in wild-type amounts under steady-state conditions (Fig. 3B). tim10–2 mitochondria are selectively impaired in the function of the Tim9-Tim10 complex (33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar, 59Frazier A.E. Chacinska A. Truscott K.N. Guiard B. Pfanner N. Rehling P. Mol. Cell. Biol. 2003; 23: 7818-7828Google Scholar). We imported the 35S-labeled precursor of Tom40 into isolated tim10–2 mitochondria. The Tom40 assembly pathway was significantly impaired at the level of the assembly intermediate I (Fig. 3C, lanes 6 and 7 versus lanes 1 and 2). To control for the specificity of the assembly defect, we used the precursor of Tom20 and found an efficient assembly reaction into the TOM complex (Fig. 3D). We conclude that both complexes of small Tim proteins of the intermembrane space, the essential Tim9-Tim10 complex and the non-essential Tim8-Tim13 complex, are involved in the assembly pathway of Tom40. In mutant mitochondria of each complex, the formation of the assembly intermediate I complex is partially inhibited. The TIM Complexes of the Intermembrane Space Are Involved in an Early Stage of the Assembly Pathway of Tom40 — Characterization of the role of small Tim proteins in the import pathway of hydrophobic inner membrane proteins indicated that these intermembrane space complexes were involved in an efficient translocation of the precursor proteins through the TOM complex to the intermembrane space (25Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Google Scholar, 26Sirrenberg C. Endres M. Fölsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Google Scholar, 28Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Google Scholar, 33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar). Tom40 inserted into the outer membrane is largely protected against externally added trypsin except for a small fragment that can be cleaved off by the protease from about half of the Tom40 molecules (16Hill K. Model K. Ryan M.T. Dietmeier K. Martin F. Wagner R. Pfanner N. Nature. 1998; 395: 516-521Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). When the Tom40 precursor has reached the SAM complex, i.e. assembly intermediate I, it already shows this characteristic protease protection (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). We used this trypsin assay to study if defects in the small Tim proteins affected an early stage of Tom40 biogenesis before formation of the assembly intermediate I. The formation of trypsin-protected Tom40 and the characteristic Tom40 fragment were indeed delayed in both tim8Δ tim13Δ mitochondria and tim10–2 mitochondria (Fig. 4, A and B). Under the same import conditions, the import and protease-protection of the matrix-targeted model preprotein Su9-DHFR, consisting of the presequence of F0-ATPase subunit 9 and the passenger dihydrofolate reductase (DHFR) (61Pfanner N. Tropschug M. Neupert W. Cell. 1987; 49: 815-823Google Scholar), were not impaired (Fig. 4, C and D). We conclude that the transfer of Tom40 precursor to a protease-protected location is delayed when small Tim proteins of the intermembrane space are impaired, indicating that small Tim proteins are involved in the assembly pathway of Tom40 before the assembly intermediate I. We asked if intermembrane space components were also required for later stages of the Tom40 assembly pathway. To selectively deplete intermembrane space components, we used the swelling assay shown in Fig. 1 at distinct import stages. Swelling led to a release of the bulk of the Tim9-Tim10 and Tim8-Tim13 complexes, as shown by Western blot analysis for Tim9 and Tim13 (Fig. 5A, lane 4), whereas proteins of the outer membrane (Tom40), inner membrane (Tim22), and matrix (citrate synthase and the cochaperone Mge1) remained completely in the mitoplast fraction (Fig. 5A, lane 3). The 35S-labeled precursor of Tom40 was accumulated at different intermediate stages and then chased to the next step of the assembly pathway (Fig. 5B; Ref. 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). Although the formation of assembly intermediate I was blocked when the mitochondria were swollen before the import reaction (Fig. 1A; Fig. 5C, lane 2), the subsequent steps were not inhibited by a swelling of mitochondria. When Tom40 precursor was first accumulated at the intermediate stage I (Fig. 5C, lane 3) and the mitochondria were then subjected to swelling, the chase to intermediate II still occurred with good efficiency (Fig. 5C, lane 5). Similarly, Tom40 accumulated at intermediate stage II (Fig. 5C, lane 6) was chased into the mature TOM complex with comparable yield in non-swollen and swollen mitochondria (Fig. 5C, lanes 7 and 8). These results indicate that a depletion of the intermembrane space selectively blocks an early stage of Tom40 assembly required for formation of the assembly intermediate I. We report that the mitochondrial intermembrane space is involved in the biogenesis pathway of the central component of the TOM complex, the protein import channel Tom40. Depletion of the intermembrane space selectively blocks the assembly pathway of Tom40 at an early stage that is required to form the assembly intermediate I (Fig. 6). Once the assembly intermediate I has been formed, i.e. the Tom40 precursor is accumulated at the SAM complex, the subsequent assembly steps are not affected by a depletion of intermembrane space components, including formation of the assembly intermediate II and the association of small Tom proteins as well as Tom receptors with Tom40 (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar). These findings have important implications for the sorting pathway of the Tom40 precursor. The Tom40 precursor accumulated at the SAM complex has been shown to be largely protected against protease added to the outside of the mitochondria, but accessible to protease upon opening of the intermembrane space by swelling (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar). Moreover, the recently identified essential subunit of the SAM complex, Sam50, contains a β-barrel domain conserved from bacteria to man (48Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Google Scholar, 49Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Google Scholar, 50Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Google Scholar). The bacterial homolog Omp85 is possibly involved in protein export to the bacterial outer membrane (62Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Google Scholar). Based on these observations it had been suggested that the precursor of Tom40, after passing through the TOM complex, may follow a conserved SAM-pathway of insertion into the outer membrane from the intermembrane space side (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 47Mihara K. Nature. 2003; 424: 505-506Google Scholar, 48Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Google Scholar, 49Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Google Scholar, 50Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Google Scholar, 63Matouschek A. Glick B.S. Nat. Struct. Biol. 2001; 8: 284-286Google Scholar). However, experimental evidence for an involvement of the intermembrane space in the assembly pathway has been lacking so far. The findings reported here indeed demonstrate that the intermembrane space plays a critical role in the assembly pathway of Tom40. The role of intermembrane space components is specific and cannot be explained by a general effect on the stability of the TOM complex because the assembly of the precursor of Tom20 into the TOM complex is not affected by depletion of intermembrane space components. We found that at least two protein complexes of the intermembrane space are involved in the assembly pathway of Tom40. Partial inactivation of the essential Tim9-Tim10 complex as well as deletion of the related, but non-essential Tim8-Tim13 complex already delayed the biogenesis pathway of Tom40 at the level of assembly intermediate I formation. The Tim9-Tim10 complex has been known so far for its role in the import of hydrophobic inner membrane carrier proteins, whereas the Tim8-Tim13 complex has been found to show a preference for the precursor of Tim23 (34Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Google Scholar, 35Davis A.J. Sepuri N.B. Holder J. Johnson A.E. Jensen R.E. J. Cell Biol. 2000; 150: 1271-1282Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar, 37Rothbauer U. Hofmann S. Mühlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Google Scholar, 38Curran S.P. Leuenberger D. Schmidt E. Koehler C.M. J. Cell Biol. 2002; 158: 1017-1027Google Scholar). These soluble TIM complexes interact with hydrophobic segments and some additional regions of the inner membrane precursor proteins and are assumed to possess chaperone-like properties in guiding the hydrophobic precursors through the aqueous intermembrane space (32Curran S.P. Leuenberger D. Oppliger W. Koehler C.M. EMBO J. 2002; 21: 942-953Google Scholar, 35Davis A.J. Sepuri N.B. Holder J. Johnson A.E. Jensen R.E. J. Cell Biol. 2000; 150: 1271-1282Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar, 38Curran S.P. Leuenberger D. Schmidt E. Koehler C.M. J. Cell Biol. 2002; 158: 1017-1027Google Scholar, 64Vial S. Lu H. Allen S. Savory P. Thornton D. Sheehan J. Tokatlidis K. J. Biol. Chem. 2002; 277: 36100-36108Google Scholar). It is thus conceivable that the complexes of these small Tim proteins assist in guiding hydrophobic segments of Tom40 exposed to the intermembrane space. Apparently, the two soluble TIM complexes can in part substitute for each other in the biogenesis pathway of Tom40, explaining why the Tom40 assembly pathway is only delayed, but not blocked when either complex is inactivated in yeast mutants. Thus the steady-state level of the TOM complex remains at a wild-type level in these mutants, excluding the concern that the observed defects in Tom40 assembly would simply be caused by a defective pre-existing TOM complex. During the import of carrier proteins and Tim23, the soluble TIM complexes promote the transfer of the precursor proteins through the TOM complex (25Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Google Scholar, 26Sirrenberg C. Endres M. Fölsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Google Scholar, 28Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Google Scholar, 33Truscott K.N. Wiedemann N. Rehling P. Müller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Google Scholar, 36Paschen S.A. Rothbauer U. Káldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Google Scholar). Similarly, we found that these TIM complexes are required for an efficient translocation of the Tom40 precursor to a protease-protected location. As recognition and translocation by the TOM complex represents the initial stage of the Tom40 import pathway (22Model K. Meisinger C. Prinz T. Wiedemann N. Truscott K.N. Pfanner N. Ryan M.T. Nat. Struct. Biol. 2001; 8: 361-370Google Scholar, 41Keil P. Weinzierl A. Kiebler M. Dietmeier K. Söllner T. Pfanner N. J. Biol. Chem. 1993; 268: 19177-19180Google Scholar, 42Rapaport D. Neupert W. J. Cell Biol. 1999; 146: 321-331Google Scholar, 46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar), we propose that the small Tim proteins and possibly further intermembrane space components are involved in the efficient transfer of the Tom40 precursor through the TOM complex to the intermembrane space side and its transfer to the SAM complex (Fig. 6). Tom40 is a β-barrel protein like the most abundant outer membrane protein porin (VDAC) (65Mannella C.A. Neuwald A.F. Lawrence C.E. J. Bioenerg. Biomembr. 1996; 28: 163-169Google Scholar, 66Blachly-Dyson E. Song J. Wolfgang W.J. Colombini M. Forte M. Mol. Cell. Biol. 1997; 17: 5727-5738Google Scholar) and the morphology protein Mdm10 of the outer membrane (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 49Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Google Scholar, 67Sogo L.F. Yaffe M.P. J. Cell Biol. 1994; 126: 1361-1373Google Scholar). The currently available evidence supports the view that the sorting and assembly pathway established for Tom40 may serve as a paradigm for the biogenesis pathways of other β-barrel proteins. The precursor of porin requires the TOM complex for the initial stages of its assembly pathway (68Krimmer T. Rapaport D. Ryan M.T. Meisinger C. Kassenbrock C.K. Blachly-Dyson E. Forte M. Douglas M.G. Neupert W. Nargang F.E. Pfanner N. J. Cell Biol. 2001; 152: 289-300Google Scholar) and rupturing of the outer membrane impairs its assembly (data not shown) (69Smith M. Hicks S. Baker K. McCauley R. J. Biol. Chem. 1994; 269: 28460-28464Google Scholar). For both porin and Mdm10 it has been shown that the SAM complex containing the highly conserved Sam50 is required for their biogenesis pathway as for Tom40 (46Wiedemann N. Kozjak V. Chacinska A. Schönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Google Scholar, 48Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Google Scholar, 49Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Google Scholar, 50Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Google Scholar). We propose that β-barrel proteins of the mitochondrial outer membrane follow a conserved biogenesis pathway which involves intermembrane space components for transfer to the SAM complex. We thank Dr. P. Rehling for the tim8Δ tim13Δ yeast mutant and A. E. Frazier and L. Sanjuán Szklarz for experimental advice." @default.
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• W1988367050 title "Biogenesis of the Protein Import Channel Tom40 of the Mitochondrial Outer Membrane" @default.
• W1988367050 cites W1507851645 @default.
• W1988367050 cites W1523656947 @default.
• W1988367050 cites W1551504358 @default.
• W1988367050 cites W1556463348 @default.
• W1988367050 cites W1577198464 @default.
• W1988367050 cites W1596172840 @default.
• W1988367050 cites W1596955355 @default.
• W1988367050 cites W1597688727 @default.
• W1988367050 cites W1600928275 @default.
• W1988367050 cites W1610607546 @default.
• W1988367050 cites W1821016817 @default.
• W1988367050 cites W1971535868 @default.
• W1988367050 cites W1978311686 @default.
• W1988367050 cites W1984378331 @default.
• W1988367050 cites W1986371684 @default.
• W1988367050 cites W1988934693 @default.
• W1988367050 cites W1989252009 @default.
• W1988367050 cites W2003874624 @default.
• W1988367050 cites W2008428619 @default.
• W1988367050 cites W2008484321 @default.
• W1988367050 cites W2013504014 @default.
• W1988367050 cites W2014889038 @default.
• W1988367050 cites W2015025581 @default.
• W1988367050 cites W2019247734 @default.
• W1988367050 cites W2023805187 @default.
• W1988367050 cites W2024144441 @default.
• W1988367050 cites W2026512409 @default.
• W1988367050 cites W2026869749 @default.
• W1988367050 cites W2028580085 @default.
• W1988367050 cites W2029471191 @default.
• W1988367050 cites W2032383870 @default.
• W1988367050 cites W2036794157 @default.
• W1988367050 cites W2037170540 @default.
• W1988367050 cites W2038758164 @default.
• W1988367050 cites W2039492407 @default.
• W1988367050 cites W2040447613 @default.
• W1988367050 cites W2047862727 @default.
• W1988367050 cites W2059492515 @default.
• W1988367050 cites W2061968489 @default.
• W1988367050 cites W2064311817 @default.
• W1988367050 cites W2069490007 @default.
• W1988367050 cites W2077307563 @default.
• W1988367050 cites W2077889625 @default.
• W1988367050 cites W2079110089 @default.
• W1988367050 cites W2081866213 @default.
• W1988367050 cites W2082516237 @default.
• W1988367050 cites W2085019401 @default.
• W1988367050 cites W2085101416 @default.
• W1988367050 cites W2107009106 @default.
• W1988367050 cites W2109859150 @default.
• W1988367050 cites W2113015426 @default.
• W1988367050 cites W2116542906 @default.
• W1988367050 cites W2119624493 @default.
• W1988367050 cites W2120245587 @default.
• W1988367050 cites W2124957811 @default.
• W1988367050 cites W2129231604 @default.
• W1988367050 cites W2131143886 @default.
• W1988367050 cites W2131630367 @default.
• W1988367050 cites W2132781144 @default.
• W1988367050 cites W2132803022 @default.
• W1988367050 cites W2137158408 @default.
• W1988367050 cites W2137418955 @default.
• W1988367050 cites W2140850020 @default.
• W1988367050 cites W2140998993 @default.
• W1988367050 cites W2151162227 @default.
• W1988367050 cites W2155006772 @default.
• W1988367050 cites W2165922683 @default.
• W1988367050 cites W2167158598 @default.
• W1988367050 cites W2169333620 @default.
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