Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Apr 2;133(2):269–280. doi: 10.1083/jcb.133.2.269

The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop

PMCID: PMC2120804  PMID: 8609161

Abstract

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adrian G. S., McCammon M. T., Montgomery D. L., Douglas M. G. Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane. Mol Cell Biol. 1986 Feb;6(2):626–634. doi: 10.1128/mcb.6.2.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassford P., Beckwith J., Ito K., Kumamoto C., Mizushima S., Oliver D., Randall L., Silhavy T., Tai P. C., Wickner B. The primary pathway of protein export in E. coli. Cell. 1991 May 3;65(3):367–368. doi: 10.1016/0092-8674(91)90453-6. [DOI] [PubMed] [Google Scholar]
  3. Berteaux-Lecellier V., Picard M., Thompson-Coffe C., Zickler D., Panvier-Adoutte A., Simonet J. M. A nonmammalian homolog of the PAF1 gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina. Cell. 1995 Jun 30;81(7):1043–1051. doi: 10.1016/s0092-8674(05)80009-1. [DOI] [PubMed] [Google Scholar]
  4. Blobel G., Walter P., Chang C. N., Goldman B. M., Erickson A. H., Lingappa V. R. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol. 1979;33:9–36. [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. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. DeGrado W. F., Wasserman Z. R., Lear J. D. Protein design, a minimalist approach. Science. 1989 Feb 3;243(4891):622–628. doi: 10.1126/science.2464850. [DOI] [PubMed] [Google Scholar]
  8. Economou A., Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 1994 Sep 9;78(5):835–843. doi: 10.1016/s0092-8674(94)90582-7. [DOI] [PubMed] [Google Scholar]
  9. Erdmann R., Blobel G. Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J Cell Biol. 1995 Feb;128(4):509–523. doi: 10.1083/jcb.128.4.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  12. Glover J. R., Andrews D. W., Rachubinski R. A. Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10541–10545. doi: 10.1073/pnas.91.22.10541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Görlich D., Rapoport T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell. 1993 Nov 19;75(4):615–630. doi: 10.1016/0092-8674(93)90483-7. [DOI] [PubMed] [Google Scholar]
  15. Hannavy K., Rospert S., Schatz G. Protein import into mitochondria: a paradigm for the translocation of polypeptides across membranes. Curr Opin Cell Biol. 1993 Aug;5(4):694–700. doi: 10.1016/0955-0674(93)90142-d. [DOI] [PubMed] [Google Scholar]
  16. Höhfeld J., Veenhuis M., Kunau W. H. PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis. J Cell Biol. 1991 Sep;114(6):1167–1178. doi: 10.1083/jcb.114.6.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jank B., Habermann B., Schweyen R. J., Link T. A. PMP47, a peroxisomal homologue of mitochondrial solute carrier proteins. Trends Biochem Sci. 1993 Nov;18(11):427–428. [PubMed] [Google Scholar]
  18. Jungnickel B., Rapoport T. A., Hartmann E. Protein translocation: common themes from bacteria to man. FEBS Lett. 1994 Jun 6;346(1):73–77. doi: 10.1016/0014-5793(94)00367-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kuan J., Saier M. H., Jr The mitochondrial carrier family of transport proteins: structural, functional, and evolutionary relationships. Crit Rev Biochem Mol Biol. 1993;28(3):209–233. doi: 10.3109/10409239309086795. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Leszczynski J. F., Rose G. D. Loops in globular proteins: a novel category of secondary structure. Science. 1986 Nov 14;234(4778):849–855. doi: 10.1126/science.3775366. [DOI] [PubMed] [Google Scholar]
  26. Liu H., Tan X., Russell K. A., Veenhuis M., Cregg J. M. PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import. J Biol Chem. 1995 May 5;270(18):10940–10951. doi: 10.1074/jbc.270.18.10940. [DOI] [PubMed] [Google Scholar]
  27. Marshall P. A., Krimkevich Y. I., Lark R. H., Dyer J. M., Veenhuis M., Goodman J. M. Pmp27 promotes peroxisomal proliferation. J Cell Biol. 1995 Apr;129(2):345–355. doi: 10.1083/jcb.129.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCammon M. T., Dowds C. A., Orth K., Moomaw C. R., Slaughter C. A., Goodman J. M. Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 25;265(33):20098–20105. [PubMed] [Google Scholar]
  29. McCammon M. T., McNew J. A., Willy P. J., Goodman J. M. An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes. J Cell Biol. 1994 Mar;124(6):915–925. doi: 10.1083/jcb.124.6.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McNew J. A., Goodman J. M. An oligomeric protein is imported into peroxisomes in vivo. J Cell Biol. 1994 Dec;127(5):1245–1257. doi: 10.1083/jcb.127.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McNew J. A., Sykes K., Goodman J. M. Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide. Mol Biol Cell. 1993 Feb;4(2):223–232. doi: 10.1091/mbc.4.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moreno M., Lark R., Campbell K. L., Goodman J. M. The peroxisomal membrane proteins of Candida boidinii: gene isolation and expression. Yeast. 1994 Nov;10(11):1447–1457. doi: 10.1002/yea.320101108. [DOI] [PubMed] [Google Scholar]
  33. Pfanner N., Hoeben P., Tropschug M., Neupert W. The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria. J Biol Chem. 1987 Nov 5;262(31):14851–14854. [PubMed] [Google Scholar]
  34. Schwarz E., Neupert W. Mitochondrial protein import: mechanisms, components and energetics. Biochim Biophys Acta. 1994 Aug 30;1187(2):270–274. doi: 10.1016/0005-2728(94)90125-2. [DOI] [PubMed] [Google Scholar]
  35. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smagula C. S., Douglas M. G. ADP-ATP carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111. J Cell Biochem. 1988 Apr;36(4):323–327. doi: 10.1002/jcb.240360402. [DOI] [PubMed] [Google Scholar]
  37. Sulter G. J., Waterham H. R., Vrieling E. G., Goodman J. M., Harder W., Veenhuis M. Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome-deficient mutant of Hansenula polymorpha. FEBS Lett. 1993 Jan 11;315(3):211–216. doi: 10.1016/0014-5793(93)81166-w. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Tan X., Waterham H. R., Veenhuis M., Cregg J. M. The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. J Cell Biol. 1995 Feb;128(3):307–319. doi: 10.1083/jcb.128.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van der Leij I., Van den Berg M., Boot R., Franse M., Distel B., Tabak H. F. Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure. J Cell Biol. 1992 Oct;119(1):153–162. doi: 10.1083/jcb.119.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Veenhuis M., Goodman J. M. Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii. J Cell Sci. 1990 Aug;96(Pt 4):583–590. doi: 10.1242/jcs.96.4.583. [DOI] [PubMed] [Google Scholar]
  43. Walton P. A., Hill P. E., Subramani S. Import of stably folded proteins into peroxisomes. Mol Biol Cell. 1995 Jun;6(6):675–683. doi: 10.1091/mbc.6.6.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Waterham H. R., Titorenko V. I., Haima P., Cregg J. M., Harder W., Veenhuis M. The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals. J Cell Biol. 1994 Nov;127(3):737–749. doi: 10.1083/jcb.127.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES