Differential Recognition of Preproteins by the Purified Cytosolic Domains of the Mitochondrial Import Receptors Tom20, Tom22, and Tom70

Abstract

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors. The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors. Nuclear-encoded mitochondrial proteins are synthesized as preproteins in the cytosol. They are targeted to receptors on the mitochondrial surface and are subsequently translocated across the outer mitochondrial membrane by a general import pore (1Kübrich M. Dietmeier K. Pfanner N. Curr. Genet. 1995; 27: 393-403Crossref PubMed Scopus (64) Google Scholar, 2Ryan K.R. Jensen R.E. Cell. 1995; 83: 517-519Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 3Lill R. Neupert W. Trends Cell. Biol. 1996; 6: 56-61Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 4Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (922) Google Scholar, 5Pfanner N. Meijer M. Curr. Biol. 1997; 7: R100-R103Abstract Full Text Full Text PDF PubMed Google Scholar). The import receptors and the general import pore assemble into a dynamic high molecular weight complex, termed the preprotein translocase of the outer mitochondrial membrane (Tom) (6Pfanner N. Douglas M.G. Endo T. Hoogenraad N.J. Jensen R.E. Meijer M. Neupert W. Schatz G. Schmitz U.K. Shore G.C. Trends Biochem. Sci. 1996; 21: 51-52Abstract Full Text PDF PubMed Scopus (118) Google Scholar). Nine Tom proteins have been identified to date. Five of them expose major portions on the cytosolic side of the outer membrane: Tom20-Tom22 and Tom70-Tom37 were suggested to function as heterodimeric import receptors for distinct classes of preproteins (see below) (7Bolliger L. Junne T. Schatz G. Lithgow T. EMBO J. 1995; 14: 6318-6326Crossref PubMed Scopus (138) Google Scholar, 8Gratzer S. Lithgow T. Bauer R.E. Lamping E. Paltauf F. Kohlwein S.D. Haucke V. Junne T. Schatz G. Horst M. J. Cell. Biol. 1995; 129: 25-34Crossref PubMed Scopus (152) Google Scholar, 9Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 3382-3389Crossref PubMed Scopus (112) Google Scholar, 10Mayer A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; 14: 4204-4211Crossref PubMed Scopus (119) Google Scholar). Tom72 is homologous to Tom70, yet is expressed at a low level and does not play a significant role in mitochondrial biogenesis (11Bömer U. Pfanner N. Dietmeier K. FEBS Lett. 1996; 382: 153-158Crossref PubMed Scopus (49) Google Scholar, 12Schlossmann J. Lill R. Neupert W. Court D.A. J. Biol. Chem. 1996; 271: 17890-17895Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The other four Tom proteins are associated with the general import pore: Tom40 is thought to represent the pore-forming component (13Vestweber D. Brunner J. Baker A. Schatz G. Nature. 1989; 341: 205-209Crossref PubMed Scopus (192) Google Scholar, 14Kiebler M. Pfaller R. Söllner T. Griffiths G. Horstmann H. Pfanner N. Neupert W. Nature. 1990; 348: 610-616Crossref PubMed Scopus (192) Google Scholar); Tom5 promotes preprotein entry into the pore (15Dietmeier, K., Hönlinger, A., Bömer, U., Dekker, P. J. T., Eckerskorn, C., Lottspeich, F., Kübrich, M., and Pfanner, N. (1997) Nature, in press.Google Scholar); and Tom6 and Tom7 modulate the association of the import pore with the receptor subcomplexes (16Alconada A. Kübrich M. Moczko M. Hönlinger A. Pfanner N. Mol. Cell. Biol. 1995; 15: 6196-6205Crossref PubMed Scopus (96) Google Scholar, 17Hönlinger A. Bömer U. Alconada A. Eckerskorn C. Lottspeich F. Dietmeier K. Pfanner N. EMBO J. 1996; 15: 2125-2137Crossref PubMed Scopus (150) Google Scholar). Protein import studies with isolated mitochondria indicated that Tom20-Tom22 functions as receptor for the typical mitochondrial preproteins that carry N-terminal cleavable targeting sequences, termed presequences, which form positively charged amphipathic α-helices (9Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 3382-3389Crossref PubMed Scopus (112) Google Scholar,18Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 19Schneider H. Söllner T. Dietmeier K. Eckerskorn C. Lottspeich F. Trülzsch B. Neupert W. Pfanner N. Science. 1991; 254: 1659-1662Crossref PubMed Scopus (87) Google Scholar, 20Ramage L. Junne T. Hahne K. Lithgow T. Schatz G. EMBO J. 1993; 12: 4115-4123Crossref PubMed Scopus (182) Google Scholar, 21Moczko M. Ehmann B. Gärtner F. Hönlinger A. Schäfer E. Pfanner N. J. Biol. Chem. 1994; 269: 9045-9051Abstract Full Text PDF PubMed Google Scholar, 22Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar). Tom70-Tom37 was reported to be required for import of noncleavable preproteins, carrying (poorly characterized) targeting information in the mature protein but also for cleavable preproteins (8Gratzer S. Lithgow T. Bauer R.E. Lamping E. Paltauf F. Kohlwein S.D. Haucke V. Junne T. Schatz G. Horst M. J. Cell. Biol. 1995; 129: 25-34Crossref PubMed Scopus (152) Google Scholar, 23Söllner T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 24Steger H.F. Söllner T. Kiebler M. Dietmeier K.A. Pfaller R. Trülzsch K.S. Tropschug M. Neupert W. Pfanner N. J. Cell. Biol. 1990; 111: 2353-2363Crossref PubMed Scopus (141) Google Scholar, 25Hines V. Brandt A. Griffiths G. Horstmann H. Brütsch H. Schatz G. EMBO J. 1990; 9: 3191-3200Crossref PubMed Scopus (203) Google Scholar, 26Hines V. Schatz G. J. Biol. Chem. 1993; 268: 449-454Abstract Full Text PDF PubMed Google Scholar). Little is known about the function and specificity of individual receptor proteins. Only the cytosolic domain of Tom70 has been purified so far and used for analyzing binding of preproteins. Schlossmann et al. (27Schlossmann J. Dietmeier K. Pfanner N. Neupert W. J. Biol. Chem. 1994; 269: 11893-11901Abstract Full Text PDF PubMed Google Scholar) showed that the cytosolic domain of Tom70 was able to bind noncleavable preproteins but also to some cleavable ones. Schleiff et al. (28Schleiff E. Shore G.C. Goping I.S. FEBS Lett. 1997; 404: 314-318Crossref PubMed Scopus (31) Google Scholar) expressed Tom20 as part of a fusion protein with glutathione S-transferase and reported the binding of various preproteins to the fusion protein but also a high degree of nonspecific binding to the beads used. For this report, we expressed and purified the cytosolic domains of Tom20, Tom22, and Tom70 and directly compared the ability of the three receptors to bind preproteins. We observed that each receptor domain could recognize and bind mitochondrial preproteins independently and with a different specificity. Only with Tom20 and Tom22, a synthetic presequence peptide competed for the binding of preproteins. Our studies indicate that each of the receptors binds mitochondrial preproteins by a different mechanism and show that two distinct presequence receptors function on the mitochondrial surface. (His)10-tagged cytosolic domains of Tom20, Tom22 and Tom70 were amplified using the polymerase chain reaction and Saccharomyces cerevisiae genomic DNA as template. To amplify yTOM20cd-His10 (N-terminal His10 tag), the primers 5′-TATCATATGGACTATCAAAGAAGAAATAGCCGCCAATTC-3′ and 5′-TATGGATCCTCAGTCATCGATATCGTTAGCTTCAGC-3′ were used. To amplify TOM70cd-His10 (N-terminal His10tag), the primers 5′-GTCGACATATGCGAGGAAAAAAGAACACGATC-3′ and 5′-GGATCCCTCGAGTTACATTAAACCCTGTTCGCG-3′ were used. The polymerase chain reaction products were digested with NdeI andBamHI or XhoI and cloned into pET19b. To amplify yTOM22cd-His10 (C-terminal His10tag), the primers 5′-TATCCATGGTCGAATTAACTGAAATTA-3′ and 5′-TATGGATCCTTAATGATGATGATGATGATGATGATGATGGTGGTTTCCGGATTTTGTGAAAGC-3′ were used. After digestion of the polymerase chain reaction product with NcoI and BamHI, it was cloned into pET19b. The correct sequences of the clones were confirmed by DNA sequencing. Escherichia coli strain BL21(DE3) was transformed with the plasmids pET19b-yTom20cd-His10, pET19b-yTom22cd-His10, or pET19b-yTom70cd-His10. The cells were grown overnight in LBA medium (0.5% (w/v) Bacto-yeast extract, 1% (w/v) Bacto-peptone, 0.5% (w/v) NaCl, 100 mg/l ampicillin) at 35 °C. 10-ml cultures were diluted 200-fold into LBA medium and grown up to anA 600 of 0.7 in a 2-liter fermenter at pH 7.0. Then isopropyl-β-d(−)-thiogalactopyranoside was added to the culture to a final concentration of 2 mm. The culture was incubated for a further 5 h. Cells were harvested by centrifugation, resuspended in 50 ml of resuspension buffer (10 mm MOPS/KOH, 1The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; DHFR, dihydrofolate reductase; PiC, phosphate carrier; Ni-NTA, nickel nitrilotriacetate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin. pH 7.2) and aliquoted. After centrifugation and removal of the supernatant, the bacterial pellets were frozen in liquid nitrogen and stored at −80 °C. The bacterial pellet was resuspended in binding buffer (5 mm imidazole, 500 mm NaCl, 20 mm Tris/HCl, pH 7.9, containing 2 mmphenylmethylsulfonyl fluoride) and sonified in a Branson sonifier at 50% duty and setting 5 with a microtip by 3 × 10 pulses of 1 s at 0 °C. The suspension was centrifuged at 16,000 ×g and 2 °C. The supernatant was applied on 1-ml Mobicol columns (Mobitec) that had been loaded with 800 μl of Ni-NTA-agarose resin slurry (Qiagen) and equilibrated with binding buffer. After an immediate spin in a microcentrifuge, the columns were washed five times with 500 μl of washing buffer (80 mm imidazole, 500 mm NaCl, 20 mm Tris/HCl, pH 7.9). The protein-loaded resin was resuspended in washing buffer and aliquoted. One aliquot was analyzed by SDS-PAGE to the and the of protein to the The of the purified proteins was confirmed by with the The protein-loaded resin of 50 of each Tom was equilibrated with buffer mm imidazole, mm 10 mm MOPS/KOH, pH 1% (w/v) by centrifugation, and the supernatant The resuspended resin was into and into 1-ml Mobicol columns that were at the A of preproteins in buffer was and the resin was resuspended by the synthetic presequence peptide or the peptide which were by and by were peptide the at of was by a The of the reaction was 100 The columns were incubated at for in a setting After centrifugation, the resin was washed three times with the buffer proteins were with buffer imidazole, 500 mm NaCl, 20 mm Tris/HCl, pH and by (w/v) After 20 of at 0 the proteins were by centrifugation and washed with The protein were analyzed by SDS-PAGE and The were as of preproteins in and with J. Biochem. PubMed Scopus Google Scholar, A. Gärtner F. Hönlinger A. Kübrich M. Pfanner N. 1995; PubMed Scopus Google Scholar) and SDS-PAGE U.K. Nature. PubMed Scopus Google Scholar). For of the cytosolic domains of S. cerevisiae Tom20, Tom22 and Tom70, the membrane of each protein was by a His10 Tom20 and Tom70 carry the membrane at the and expose the on the cytosolic side H. Söllner T. Dietmeier K. Eckerskorn C. Lottspeich F. Trülzsch B. Neupert W. Pfanner N. Science. 1991; 254: 1659-1662Crossref PubMed Scopus (87) Google Scholar, 24Steger H.F. Söllner T. Kiebler M. Dietmeier K.A. Pfaller R. Trülzsch K.S. Tropschug M. Neupert W. Pfanner N. J. Cell. Biol. 1990; 111: 2353-2363Crossref PubMed Scopus (141) Google Scholar, 25Hines V. Brandt A. Griffiths G. Horstmann H. Brütsch H. Schatz G. EMBO J. 1990; 9: 3191-3200Crossref PubMed Scopus (203) Google Scholar); the His10 was at the of the cytosolic Tom22 the on the cytosolic side and a membrane in the and a on the side M. P. H. Pfanner N. Neupert W. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). The cytosolic domain of Tom22 was with a His10 of the and by the cytosolic domains were expressed in E. coli cells to of protein 1 The cytosolic domains were purified the of coli cells by The proteins were 1 binding of mitochondrial preproteins to purified receptor of the cytosolic domains of Tom20, Tom22, and Tom70 were to For the the of coli strain was to a with Then an preproteins in buffer 100 mm was at for as and After removal of the the protein were and analyzed by was of preprotein that was added to the was by The of each preprotein added was to 1 and of the cytosolic domains of Tom20, Tom22, and Tom70. of Tom20, Tom22, and Tom70. outer mitochondrial in E. of the cytosolic of Tom20, of Tom22, and of Tom70 containing a His10 of the membrane was by isopropyl-β-d(−)-thiogalactopyranoside as and The cells were by and aliquot of the supernatant was by SDS-PAGE and by The protein of a strain is in molecular The of and are with in and purified cytosolic The proteins were purified by The was analyzed by SDS-PAGE and with The of and was the of the cytosolic domains of Tom20, Tom22, and Tom70 to the resin were used for binding with mitochondrial preproteins The resin that was to a with E. coli cells The preproteins were synthesized in in the of a fusion protein the presequence of and dihydrofolate N. Tropschug M. Neupert W. Cell. Full Text PDF PubMed Scopus (192) Google Scholar); as the cytosolic a fusion protein containing the presequence N-terminal of mature 2 and the a of the presequence was H. B. J. J. Neupert W. Cell. Full Text PDF PubMed Scopus Google Scholar); the of 1 that an N-terminal presequence with a and a membrane in the mature protein the phosphate that is synthesized a presequence but internal targeting and membrane The preproteins were incubated with the cytosolic domains at After proteins were and analyzed by SDS-PAGE and Each of the cytosolic domains with a distinct of the proteins four mitochondrial preproteins preferentially and 1 and binding of was in the 2 and 1 and but not the fusion proteins not bind to of the three cytosolic domains To binding of preproteins to the cytosolic we a of the purified cytosolic in the binding the of not the and of preprotein binding We conclude that the cytosolic domains of Tom20, Tom22, and Tom70 selectively bind distinct of mitochondrial preproteins. The cytosolic domain of Tom22 a of charged and was suggested to with the positively charged amphipathic presequences (7Bolliger L. Junne T. Schatz G. Lithgow T. EMBO J. 1995; 14: 6318-6326Crossref PubMed Scopus (138) Google Scholar, 9Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 3382-3389Crossref PubMed Scopus (112) Google Scholar, 10Mayer A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; 14: 4204-4211Crossref PubMed Scopus (119) Google Scholar, M. P. H. Pfanner N. Neupert W. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). We to with a low The binding reported in 2 were in the of 100 mm that could with a the charged of presequences and low the binding of to enhanced the binding of to was at low that of 1 was and not bind to at concentration Tom20 a with charged and was suggested to bind preproteins by (7Bolliger L. Junne T. Schatz G. Lithgow T. EMBO J. 1995; 14: 6318-6326Crossref PubMed Scopus (138) Google Scholar, 10Mayer A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; 14: 4204-4211Crossref PubMed Scopus (119) Google M. Ehmann B. Gärtner F. Hönlinger A. Schäfer E. Pfanner N. J. Biol. Chem. 1994; 269: 9045-9051Abstract Full Text PDF PubMed Google Scholar, V. Lithgow T. S. Hahne K. Schatz G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). We of the concentration enhanced preprotein binding to the binding of and as as that of 1 and to at low in the concentration of to mm enhanced binding of four preproteins to not bind at concentration The binding of preproteins to and an on the concentration of 1 and to were by a of the concentration mm and not bind to at We conclude that binding of preproteins to is by the binding of preproteins to is by salt. of preproteins to is Only is able to bind significant of four mitochondrial preproteins To further the specificity of preprotein binding to the cytosolic we used two chemically synthesized The N-terminal of the presequence of of function as a mitochondrial import form an amphipathic with a positively charged side and a side Schatz G. Proc. Natl. Acad. Sci. U. S. A. 83: PubMed Scopus Google Scholar, D. Schatz G. EMBO J. PubMed Scopus Google Scholar, D. F. Schatz G. EMBO J. 7: PubMed Scopus Google Scholar, FEBS Lett. 1990; PubMed Scopus Google Scholar). The peptide the of positively charged but the and does not function as mitochondrial import Schatz G. Proc. Natl. Acad. Sci. U. S. A. 83: PubMed Scopus Google Scholar). of preproteins to was in the of different of the presequence peptide or the The presequence peptide the binding of and 5 of preprotein binding at peptide in the of and at peptide in the of up to 20 the peptide a on binding of two preproteins to and and at it a of binding and The of the presequence peptide on binding of 1 to was not as as that on the binding of the two fusion proteins but distinct that of the peptide and of the preproteins 1 and to was by the presequence peptide by the peptide independently of the concentration in the the presequence peptide competed with the binding of three preproteins, and in to the peptide The of binding was at a concentration of the presequence peptide of in the of and and in the of the of was A and of the noncleavable preprotein to was not competed for by the presequence peptide and We conclude that a chemically synthesized presequence peptide competes with the binding of cleavable preproteins to and of preproteins to is not by the presequence We that the purified cytosolic domains of Tom20, Tom22, and Tom70 selectively bind distinct of mitochondrial preproteins. The of preprotein binding to the domains with the preprotein that have been for Tom proteins by function with mitochondria or outer membrane in M. Dietmeier K. Pfanner N. Curr. Genet. 1995; 27: 393-403Crossref PubMed Scopus (64) Google Scholar, 3Lill R. Neupert W. Trends Cell. Biol. 1996; 6: 56-61Abstract Full Text PDF PubMed Scopus (116) Google Scholar, and T. Schatz G. Trends Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). with the of binding of a protein and the by a synthetic presequence this the of the in binding The in the purified in the mechanism of of mitochondrial import receptors. each cytosolic domain bind preproteins a for other Tom proteins. the of binding of the preprotein are different for each of the cytosolic different for the three import receptors. it has been that Tom70, which preferentially binds preproteins with internal targeting information T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 24Steger H.F. Söllner T. Kiebler M. Dietmeier K.A. Pfaller R. Trülzsch K.S. Tropschug M. Neupert W. Pfanner N. J. Cell. Biol. 1990; 111: 2353-2363Crossref PubMed Scopus (141) Google Scholar, 25Hines V. Brandt A. Griffiths G. Horstmann H. Brütsch H. Schatz G. EMBO J. 1990; 9: 3191-3200Crossref PubMed Scopus (203) Google Scholar, 26Hines V. Schatz G. J. Biol. Chem. 1993; 268: 449-454Abstract Full Text PDF PubMed Google Scholar, J. Dietmeier K. Pfanner N. Neupert W. J. Biol. Chem. 1994; 269: 11893-11901Abstract Full Text PDF PubMed Google functions Tom20 and Tom22, the two receptors were to bind preproteins by a mechanism. Both were suggested to bind positively charged presequences in a salt-sensitive (7Bolliger L. Junne T. Schatz G. Lithgow T. EMBO J. 1995; 14: 6318-6326Crossref PubMed Scopus (138) Google Scholar, 9Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 3382-3389Crossref PubMed Scopus (112) Google Scholar, 10Mayer A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; 14: 4204-4211Crossref PubMed Scopus (119) Google Scholar, 21Moczko M. Ehmann B. Gärtner F. Hönlinger A. Schäfer E. Pfanner N. J. Biol. Chem. 1994; 269: 9045-9051Abstract Full Text PDF PubMed Google Scholar, V. Lithgow T. S. Hahne K. Schatz G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). al. A. Nargang F.E. Neupert W. Lill R. EMBO J. 1995; 14: 4204-4211Crossref PubMed Scopus (119) Google Scholar) that Tom20 and Tom22 not binding for preproteins but function as a heterodimeric The reported the of mitochondrial import receptors. to the of a cleavable of a which of an amphipathic mitochondrial presequence and a cytosolic is to the addition of and is competed for by a synthetic presequence that binding the presequence and that the positively charged surface of the presequence with the charged M. P. H. Pfanner N. Neupert W. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar) in a manner. and noncleavable preproteins bind to a specificity for this import receptor in with the binding of a fusion protein and Tom20 (28Schleiff E. Shore G.C. Goping I.S. FEBS Lett. 1997; 404: 314-318Crossref PubMed Scopus (31) Google Scholar). The binding of preproteins to is by the addition of to the that preprotein recognition by is not by but by a of with the surface of the amphipathic a concentration of a which the of as the presequence peptide but the preprotein binding to but not to The of the that and recognize distinct of a has been that synthetic presequences form amphipathic α-helices at as or but not in D. Schatz G. EMBO J. PubMed Scopus Google Scholar, D. F. Schatz G. EMBO J. 7: PubMed Scopus Google Scholar, FEBS Lett. 1990; PubMed Scopus Google that the of presequences with the binding of a receptor the of an a synthetic presequence peptide competes with the binding of cleavable preproteins but not with that of a noncleavable to it is that Tom20 distinct for recognition of presequences and internal targeting of preproteins to to the or the of and is not competed for by the presequence the binding that of and does not recognize mitochondrial presequences but targeting information in the mature part of preproteins. The cleavable preprotein 1 other cleavable preproteins it does not bind to and but also to receptor of 1 is in with import studies of this preprotein into mitochondria V. Schatz G. J. Biol. Chem. 1993; 268: 449-454Abstract Full Text PDF PubMed Google Scholar, A. P. Pfanner N. Biol. Chem. 1995; Scholar). the binding to and is competed for by the synthetic presequence and the binding to is by the that not carry targeting information the amphipathic part of the presequence but also in protein further is to internal targeting of studies on the import of preproteins into mitochondria suggested that some cleavable preproteins carry targeting information not in the presequence but also the mature protein S.D. Mol. Cell. Biol. 7: PubMed Scopus Google Scholar, N. Neupert W. EMBO J. 6: PubMed Scopus Google Scholar, V. F. K. Pfanner N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The binding observed with different preproteins the that as as a binding for preproteins The major binding of is that for the positively charged surface of internal targeting information of a preprotein the binding but is not to binding by A binding on not function independently but play an two binding for the surface of presequences and for internal targeting each of which is for recognition of preproteins. a binding for internal targeting information that is not by of it is that each of the three import receptors Tom20, Tom22, and Tom70 bind mitochondrial preproteins independently of other Tom proteins. this that two presequence receptors at the mitochondrial outer The concentration of presequence peptide for of binding a low for the import receptors. The of two presequence receptors that recognize distinct of the presequence the specificity and of preprotein recognition at the mitochondrial surface. We for plasmids and for on the