Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Feb 1;132(3):325–334. doi: 10.1083/jcb.132.3.325

Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide

PMCID: PMC2120724  PMID: 8636211

Abstract

Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16- amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Bartel P., Chien C. T., Sternglanz R., Fields S. Elimination of false positives that arise in using the two-hybrid system. Biotechniques. 1993 Jun;14(6):920–924. [PubMed] [Google Scholar]
  2. Baudhuin P., Beaufay H., Rahman-Li Y., Sellinger O. Z., Wattiaux R., Jacques P., De Duve C. Tissue fractionation studies. 17. Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue. Biochem J. 1964 Jul;92(1):179–184. doi: 10.1042/bj0920179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blattner J., Dörsam H., Clatyon C. E. Function of N-terminal import signals in trypanosome microbodies. FEBS Lett. 1995 Mar 6;360(3):310–314. doi: 10.1016/0014-5793(95)00128-v. [DOI] [PubMed] [Google Scholar]
  4. Brocard C., Kragler F., Simon M. M., Schuster T., Hartig A. The tetratricopeptide repeat-domain of the PAS10 protein of Saccharomyces cerevisiae is essential for binding the peroxisomal targeting signal-SKL. Biochem Biophys Res Commun. 1994 Nov 15;204(3):1016–1022. doi: 10.1006/bbrc.1994.2564. [DOI] [PubMed] [Google Scholar]
  5. Chevray P. M., Nathans D. Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5789–5793. doi: 10.1073/pnas.89.13.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dodt G., Braverman N., Wong C., Moser A., Moser H. W., Watkins P., Valle D., Gould S. J. Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet. 1995 Feb;9(2):115–125. doi: 10.1038/ng0295-115. [DOI] [PubMed] [Google Scholar]
  7. Erdmann R., Kunau W. H. Purification and immunolocalization of the peroxisomal 3-oxoacyl-CoA thiolase from Saccharomyces cerevisiae. Yeast. 1994 Sep;10(9):1173–1182. doi: 10.1002/yea.320100905. [DOI] [PubMed] [Google Scholar]
  8. Erdmann R. The peroxisomal targeting signal of 3-oxoacyl-CoA thiolase from Saccharomyces cerevisiae. Yeast. 1994 Jul;10(7):935–944. doi: 10.1002/yea.320100708. [DOI] [PubMed] [Google Scholar]
  9. Faber K. N., Keizer-Gunnink I., Pluim D., Harder W., Ab G., Veenhuis M. The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEBS Lett. 1995 Jan 3;357(2):115–120. doi: 10.1016/0014-5793(94)01317-t. [DOI] [PubMed] [Google Scholar]
  10. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  11. Fransen M., Brees C., Baumgart E., Vanhooren J. C., Baes M., Mannaerts G. P., Van Veldhoven P. P. Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor. J Biol Chem. 1995 Mar 31;270(13):7731–7736. doi: 10.1074/jbc.270.13.7731. [DOI] [PubMed] [Google Scholar]
  12. Gietl C. Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5773–5777. doi: 10.1073/pnas.87.15.5773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Glover J. R., Andrews D. W., Subramani S., Rachubinski R. A. Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo. J Biol Chem. 1994 Mar 11;269(10):7558–7563. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  18. Igual J. C., Matallaná E., Gonzalez-Bosch C., Franco L., Pérez-Ortin J. E. A new glucose-repressible gene identified from the analysis of chromatin structure in deletion mutants of yeast SUC2 locus. Yeast. 1991 May-Jun;7(4):379–389. doi: 10.1002/yea.320070408. [DOI] [PubMed] [Google Scholar]
  19. Kang P. J., Ostermann J., Shilling J., Neupert W., Craig E. A., Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990 Nov 8;348(6297):137–143. doi: 10.1038/348137a0. [DOI] [PubMed] [Google Scholar]
  20. Keller G. A., Krisans S., Gould S. J., Sommer J. M., Wang C. C., Schliebs W., Kunau W., Brody S., Subramani S. Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J Cell Biol. 1991 Sep;114(5):893–904. doi: 10.1083/jcb.114.5.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kragler F., Langeder A., Raupachova J., Binder M., Hartig A. Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae. J Cell Biol. 1993 Feb;120(3):665–673. doi: 10.1083/jcb.120.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kunau W. H., Beyer A., Franken T., Götte K., Marzioch M., Saidowsky J., Skaletz-Rorowski A., Wiebel F. F. Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics. Biochimie. 1993;75(3-4):209–224. doi: 10.1016/0300-9084(93)90079-8. [DOI] [PubMed] [Google Scholar]
  23. Lazarow P. B. Genetic approaches to studying peroxisome biogenesis. Trends Cell Biol. 1993 Mar;3(3):89–93. doi: 10.1016/0962-8924(93)90079-g. [DOI] [PubMed] [Google Scholar]
  24. Lazarow P. B., Thieringer R., Cohen G., Imanaka T., Small G. Protein import into peroxisomes in vitro. Methods Cell Biol. 1991;34:303–326. doi: 10.1016/s0091-679x(08)61687-8. [DOI] [PubMed] [Google Scholar]
  25. Marzioch M., Erdmann R., Veenhuis M., Kunau W. H. PAS7 encodes a novel yeast member of the WD-40 protein family essential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes. EMBO J. 1994 Oct 17;13(20):4908–4918. doi: 10.1002/j.1460-2075.1994.tb06818.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Motley A., Hettema E., Distel B., Tabak H. Differential protein import deficiencies in human peroxisome assembly disorders. J Cell Biol. 1994 May;125(4):755–767. doi: 10.1083/jcb.125.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Neer E. J., Schmidt C. J., Nambudripad R., Smith T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994 Sep 22;371(6495):297–300. doi: 10.1038/371297a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Poole B., Leighton F., De Duve C. The synthesis and turnover of rat liver peroxisomes. II. Turnover of peroxisome proteins. J Cell Biol. 1969 May;41(2):536–546. doi: 10.1083/jcb.41.2.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Purdue P. E., Lazarow P. B. Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes. Yeast. 1995 Sep 15;11(11):1045–1060. doi: 10.1002/yea.320111106. [DOI] [PubMed] [Google Scholar]
  32. Purdue P. E., Lazarow P. B. Peroxisomal biogenesis: multiple pathways of protein import. J Biol Chem. 1994 Dec 2;269(48):30065–30068. [PubMed] [Google Scholar]
  33. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  34. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  35. Shimozawa N., Tsukamoto T., Suzuki Y., Orii T., Shirayoshi Y., Mori T., Fujiki Y. A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science. 1992 Feb 28;255(5048):1132–1134. doi: 10.1126/science.1546315. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Small G. M., Szabo L. J., Lazarow P. B. Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. EMBO J. 1988 Apr;7(4):1167–1173. doi: 10.1002/j.1460-2075.1988.tb02927.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Subramani S. Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol. 1993;9:445–478. doi: 10.1146/annurev.cb.09.110193.002305. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Thieringer R., Shio H., Han Y. S., Cohen G., Lazarow P. B. Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes. Mol Cell Biol. 1991 Jan;11(1):510–522. doi: 10.1128/mcb.11.1.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van der Leij I., Franse M. M., Elgersma Y., Distel B., Tabak H. F. PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11782–11786. doi: 10.1073/pnas.90.24.11782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Wiemer E. A., Nuttley W. M., Bertolaet B. L., Li X., Francke U., Wheelock M. J., Anné U. K., Johnson K. R., Subramani S. Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J Cell Biol. 1995 Jul;130(1):51–65. doi: 10.1083/jcb.130.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang J. W., Han Y., Lazarow P. B. Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae. J Cell Biol. 1993 Dec;123(5):1133–1147. doi: 10.1083/jcb.123.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang J. W., Lazarow P. B. PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra-peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes. J Cell Biol. 1995 Apr;129(1):65–80. doi: 10.1083/jcb.129.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang J. W., Luckey C., Lazarow P. B. Three peroxisome protein packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis. Mol Biol Cell. 1993 Dec;4(12):1351–1359. doi: 10.1091/mbc.4.12.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES