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. 1993 Dec 2;123(6):1717–1725. doi: 10.1083/jcb.123.6.1717

In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes

PMCID: PMC2290900  PMID: 8276892

Abstract

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.

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Selected References

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  1. Ahnert-Hilger G., Mach W., Föhr K. J., Gratzl M. Poration by alpha-toxin and streptolysin O: an approach to analyze intracellular processes. Methods Cell Biol. 1989;31:63–90. doi: 10.1016/s0091-679x(08)61602-7. [DOI] [PubMed] [Google Scholar]
  2. Barnard E. A. Receptor classes and the transmitter-gated ion channels. Trends Biochem Sci. 1992 Oct;17(10):368–374. doi: 10.1016/0968-0004(92)90002-q. [DOI] [PubMed] [Google Scholar]
  3. Bhakdi S., Tranum-Jensen J., Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infect Immun. 1985 Jan;47(1):52–60. doi: 10.1128/iai.47.1.52-60.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bodnar A. G., Rachubinski R. A. Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats. Biochem Cell Biol. 1991 Aug;69(8):499–508. doi: 10.1139/o91-074. [DOI] [PubMed] [Google Scholar]
  6. Borst P. Peroxisome biogenesis revisited. Biochim Biophys Acta. 1989 Jun 1;1008(1):1–13. doi: 10.1016/0167-4781(89)90163-2. [DOI] [PubMed] [Google Scholar]
  7. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  8. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  9. Ellis R. J., Hemmingsen S. M. Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures. Trends Biochem Sci. 1989 Aug;14(8):339–342. doi: 10.1016/0968-0004(89)90168-0. [DOI] [PubMed] [Google Scholar]
  10. Fujiki Y., Fowler S., Shio H., Hubbard A. L., Lazarow P. B. Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J Cell Biol. 1982 Apr;93(1):103–110. doi: 10.1083/jcb.93.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujiki Y., Lazarow P. B. Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro. J Biol Chem. 1985 May 10;260(9):5603–5609. [PubMed] [Google Scholar]
  12. Fujiki Y., Rachubinski R. A., Lazarow P. B. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7127–7131. doi: 10.1073/pnas.81.22.7127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Galzi J. L., Revah F., Bessis A., Changeux J. P. Functional architecture of the nicotinic acetylcholine receptor: from electric organ to brain. Annu Rev Pharmacol Toxicol. 1991;31:37–72. doi: 10.1146/annurev.pa.31.040191.000345. [DOI] [PubMed] [Google Scholar]
  14. Goldman B. M., Blobel G. Biogenesis of peroxisomes: intracellular site of synthesis of catalase and uricase. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5066–5070. doi: 10.1073/pnas.75.10.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gould S. G., Keller G. A., Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol. 1987 Dec;105(6 Pt 2):2923–2931. doi: 10.1083/jcb.105.6.2923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gould S. J., Keller G. A., Subramani S. Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol. 1988 Sep;107(3):897–905. doi: 10.1083/jcb.107.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gould S. J., Krisans S., Keller G. A., Subramani S. Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins. J Cell Biol. 1990 Jan;110(1):27–34. doi: 10.1083/jcb.110.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hartl F. U., Just W. W. Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states. Arch Biochem Biophys. 1987 May 15;255(1):109–119. doi: 10.1016/0003-9861(87)90300-6. [DOI] [PubMed] [Google Scholar]
  20. Hartl F. U., Just W. W., Köster A., Schimassek H. Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation. Arch Biochem Biophys. 1985 Feb 15;237(1):124–134. doi: 10.1016/0003-9861(85)90261-9. [DOI] [PubMed] [Google Scholar]
  21. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  22. Hashimoto T., Kuwabara T., Usuda N., Nagata T. Purification of membrane polypeptides of rat liver peroxisomes. J Biochem. 1986 Aug;100(2):301–310. doi: 10.1093/oxfordjournals.jbchem.a121716. [DOI] [PubMed] [Google Scholar]
  23. Heinemann P., Just W. W. Peroxisomal protein import. In vivo evidence for a novel translocation competent compartment. FEBS Lett. 1992 Mar 30;300(2):179–182. doi: 10.1016/0014-5793(92)80191-i. [DOI] [PubMed] [Google Scholar]
  24. Imanaka T., Small G. M., Lazarow P. B. Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential. J Cell Biol. 1987 Dec;105(6 Pt 2):2915–2922. doi: 10.1083/jcb.105.6.2915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jakob U., Gaestel M., Engel K., Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem. 1993 Jan 25;268(3):1517–1520. [PubMed] [Google Scholar]
  26. Just W. W., Gorgas K., Hartl F. U., Heinemann P., Salzer M., Schimassek H. Biochemical effects and zonal heterogeneity of peroxisome proliferation induced by perfluorocarboxylic acids in rat liver. Hepatology. 1989 Apr;9(4):570–581. doi: 10.1002/hep.1840090411. [DOI] [PubMed] [Google Scholar]
  27. Just W. W., Soto U. Biogenesis of peroxisomes in mammals. Cell Biochem Funct. 1992 Sep;10(3):159–165. doi: 10.1002/cbf.290100305. [DOI] [PubMed] [Google Scholar]
  28. Kaldi K., Diestelkötter P., Stenbeck G., Auerbach S., Jäkle U., Mägert H. J., Wieland F. T., Just W. W. Membrane topology of the 22 kDa integral peroxisomal membrane protein. FEBS Lett. 1993 Jan 11;315(3):217–222. doi: 10.1016/0014-5793(93)81167-x. [DOI] [PubMed] [Google Scholar]
  29. Kamijo K., Taketani S., Yokota S., Osumi T., Hashimoto T. The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily. J Biol Chem. 1990 Mar 15;265(8):4534–4540. [PubMed] [Google Scholar]
  30. Lazarow P. B., Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol. 1985;1:489–530. doi: 10.1146/annurev.cb.01.110185.002421. [DOI] [PubMed] [Google Scholar]
  31. Lundin A., Hasenson M., Persson J., Pousette A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 1986;133:27–42. doi: 10.1016/0076-6879(86)33053-2. [DOI] [PubMed] [Google Scholar]
  32. McCollum D., Monosov E., Subramani S. The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family. J Cell Biol. 1993 May;121(4):761–774. doi: 10.1083/jcb.121.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Merck K. B., Groenen P. J., Voorter C. E., de Haard-Hoekman W. A., Horwitz J., Bloemendal H., de Jong W. W. Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones. J Biol Chem. 1993 Jan 15;268(2):1046–1052. [PubMed] [Google Scholar]
  34. Miura S., Mori M., Takiguchi M., Tatibana M., Furuta S., Miyazawa S., Hashimoto T. Biosynthesis and intracellular transport of enzymes of peroxisomal beta-oxidation. J Biol Chem. 1984 May 25;259(10):6397–6402. [PubMed] [Google Scholar]
  35. Murakami H., Pain D., Blobel G. 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J Cell Biol. 1988 Dec;107(6 Pt 1):2051–2057. doi: 10.1083/jcb.107.6.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Newmeyer D. D., Forbes D. J. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. doi: 10.1083/jcb.110.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Osumi T., Tsukamoto T., Hata S., Yokota S., Miura S., Fujiki Y., Hijikata M., Miyazawa S., Hashimoto T. Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Commun. 1991 Dec 31;181(3):947–954. doi: 10.1016/0006-291x(91)92028-i. [DOI] [PubMed] [Google Scholar]
  38. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  39. Pfanner N., Neupert W. The mitochondrial protein import apparatus. Annu Rev Biochem. 1990;59:331–353. doi: 10.1146/annurev.bi.59.070190.001555. [DOI] [PubMed] [Google Scholar]
  40. Pfanner N., Neupert W. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986 Dec 15;209(2):152–156. doi: 10.1016/0014-5793(86)81101-2. [DOI] [PubMed] [Google Scholar]
  41. Rapoport T. A. Protein transport across the ER membrane. Trends Biochem Sci. 1990 Sep;15(9):355–358. doi: 10.1016/0968-0004(90)90076-n. [DOI] [PubMed] [Google Scholar]
  42. Rapp S., Soto U., Just W. W. Import of firefly luciferase into peroxisomes of permeabilized Chinese hamster ovary cells: a model system to study peroxisomal protein import in vitro. Exp Cell Res. 1993 Mar;205(1):59–65. doi: 10.1006/excr.1993.1058. [DOI] [PubMed] [Google Scholar]
  43. Robbi M., Lazarow P. B. Synthesis of catalase in two cell-free protein-synthesizing systems and in rat liver. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4344–4348. doi: 10.1073/pnas.75.9.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  45. Sheffield W. P., Shore G. C., Randall S. K. Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence. J Biol Chem. 1990 Jul 5;265(19):11069–11076. [PubMed] [Google Scholar]
  46. Siegel V., Walter P. Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988 Jan 15;52(1):39–49. doi: 10.1016/0092-8674(88)90529-6. [DOI] [PubMed] [Google Scholar]
  47. Simon S. M., Peskin C. S., Oster G. F. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. doi: 10.1073/pnas.89.9.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Singer S. J. The structure and insertion of integral proteins in membranes. Annu Rev Cell Biol. 1990;6:247–296. doi: 10.1146/annurev.cb.06.110190.001335. [DOI] [PubMed] [Google Scholar]
  49. Soto U., Pepperkok R., Ansorge W., Just W. W. Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates. Exp Cell Res. 1993 Mar;205(1):66–75. doi: 10.1006/excr.1993.1059. [DOI] [PubMed] [Google Scholar]
  50. Suzuki Y., Orii T., Takiguchi M., Mori M., Hijikata M., Hashimoto T. Biosynthesis of membrane polypeptides of rat liver peroxisomes. J Biochem. 1987 Feb;101(2):491–496. doi: 10.1093/oxfordjournals.jbchem.a121935. [DOI] [PubMed] [Google Scholar]
  51. Swinkels B. W., Gould S. J., Bodnar A. G., Rachubinski R. A., Subramani S. A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J. 1991 Nov;10(11):3255–3262. doi: 10.1002/j.1460-2075.1991.tb04889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Verner K., Schatz G. Protein translocation across membranes. Science. 1988 Sep 9;241(4871):1307–1313. doi: 10.1126/science.2842866. [DOI] [PubMed] [Google Scholar]
  53. Weiher H., Noda T., Gray D. A., Sharpe A. H., Jaenisch R. Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome. Cell. 1990 Aug 10;62(3):425–434. doi: 10.1016/0092-8674(90)90008-3. [DOI] [PubMed] [Google Scholar]
  54. Wendland M., Subramani S. Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J Cell Biol. 1993 Feb;120(3):675–685. doi: 10.1083/jcb.120.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wiech H., Buchner J., Zimmermann R., Jakob U. Hsp90 chaperones protein folding in vitro. Nature. 1992 Jul 9;358(6382):169–170. doi: 10.1038/358169a0. [DOI] [PubMed] [Google Scholar]

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