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. 1996 Jun 17;15(12):2901–2913.

The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene.

P Rehling 1, M Marzioch 1, F Niesen 1, E Wittke 1, M Veenhuis 1, W H Kunau 1
PMCID: PMC450230  PMID: 8670791

Abstract

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein beta-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired.

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Selected References

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

  1. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  2. Bout A., Teunissen Y., Hashimoto T., Benne R., Tager J. M. Nucleotide sequence of human peroxisomal 3-oxoacyl-CoA thiolase. Nucleic Acids Res. 1988 Nov 11;16(21):10369–10369. doi: 10.1093/nar/16.21.10369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Cartwright C. P., Li Y., Zhu Y. S., Kang Y. S., Tipper D. J. Use of beta-lactamase as a secreted reporter of promoter function in yeast. Yeast. 1994 Apr;10(4):497–508. doi: 10.1002/yea.320100409. [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. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crane D. I., Kalish J. E., Gould S. J. The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly. J Biol Chem. 1994 Aug 26;269(34):21835–21844. [PubMed] [Google Scholar]
  8. 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]
  9. Einerhand A. W., Voorn-Brouwer T. M., Erdmann R., Kunau W. H., Tabak H. F. Regulation of transcription of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase of Saccharomyces cerevisiae. Eur J Biochem. 1991 Aug 15;200(1):113–122. doi: 10.1111/j.1432-1033.1991.tb21056.x. [DOI] [PubMed] [Google Scholar]
  10. Elgersma Y., van Roermund C. W., Wanders R. J., Tabak H. F. Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene. EMBO J. 1995 Jul 17;14(14):3472–3479. doi: 10.1002/j.1460-2075.1995.tb07353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elgersma Y., van den Berg M., Tabak H. F., Distel B. An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae. Genetics. 1993 Nov;135(3):731–740. doi: 10.1093/genetics/135.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Gietl C., Faber K. N., van der Klei I. J., Veenhuis M. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3151–3155. doi: 10.1073/pnas.91.8.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Goebl M., Yanagida M. The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci. 1991 May;16(5):173–177. doi: 10.1016/0968-0004(91)90070-c. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Gould S. J., Keller G. A., Schneider M., Howell S. H., Garrard L. J., Goodman J. M., Distel B., Tabak H., Subramani S. Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J. 1990 Jan;9(1):85–90. doi: 10.1002/j.1460-2075.1990.tb08083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hachiya N., Komiya T., Alam R., Iwahashi J., Sakaguchi M., Omura T., Mihara K. MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. EMBO J. 1994 Nov 1;13(21):5146–5154. doi: 10.1002/j.1460-2075.1994.tb06844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. Kurzchalia T. V., Wiedmann M., Girshovich A. S., Bochkareva E. S., Bielka H., Rapoport T. A. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. doi: 10.1038/320634a0. [DOI] [PubMed] [Google Scholar]
  30. Lamb J. R., Michaud W. A., Sikorski R. S., Hieter P. A. Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J. 1994 Sep 15;13(18):4321–4328. doi: 10.1002/j.1460-2075.1994.tb06752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamb J. R., Tugendreich S., Hieter P. Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem Sci. 1995 Jul;20(7):257–259. doi: 10.1016/s0968-0004(00)89037-4. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Mauri I., Maddaloni M., Lohmer S., Motto M., Salamini F., Thompson R., Martegani E. Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast. Mol Gen Genet. 1993 Nov;241(3-4):319–326. doi: 10.1007/BF00284684. [DOI] [PubMed] [Google Scholar]
  34. McCammon M. T., Veenhuis M., Trapp S. B., Goodman J. M. Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J Bacteriol. 1990 Oct;172(10):5816–5827. doi: 10.1128/jb.172.10.5816-5827.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Moreno de la Garza M., Schultz-Borchard U., Crabb J. W., Kunau W. H. Peroxisomal beta-oxidation system of Candida tropicalis. Purification of a multifunctional protein possessing enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities. Eur J Biochem. 1985 Apr 15;148(2):285–291. doi: 10.1111/j.1432-1033.1985.tb08837.x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Murakami K., Mori M. Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein. EMBO J. 1990 Oct;9(10):3201–3208. doi: 10.1002/j.1460-2075.1990.tb07518.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Nuttley W. M., Szilard R. K., Smith J. J., Veenhuis M., Rachubinski R. A. The PAH2 gene is required for peroxisome assembly in the methylotrophic yeast Hansenula polymorpha and encodes a member of the tetratricopeptide repeat family of proteins. Gene. 1995 Jul 4;160(1):33–39. doi: 10.1016/0378-1119(95)00230-4. [DOI] [PubMed] [Google Scholar]
  42. Powers M. A., Forbes D. J. Cytosolic factors in nuclear transport: what's importin? Cell. 1994 Dec 16;79(6):931–934. doi: 10.1016/0092-8674(94)90024-8. [DOI] [PubMed] [Google Scholar]
  43. Randall L. L., Hardy S. J. High selectivity with low specificity: how SecB has solved the paradox of chaperone binding. Trends Biochem Sci. 1995 Feb;20(2):65–69. doi: 10.1016/s0968-0004(00)88959-8. [DOI] [PubMed] [Google Scholar]
  44. Slawecki M. L., Dodt G., Steinberg S., Moser A. B., Moser H. W., Gould S. J. Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders. J Cell Sci. 1995 May;108(Pt 5):1817–1829. doi: 10.1242/jcs.108.5.1817. [DOI] [PubMed] [Google Scholar]
  45. Stanfield R. L., Wilson I. A. Protein-peptide interactions. Curr Opin Struct Biol. 1995 Feb;5(1):103–113. doi: 10.1016/0959-440x(95)80015-s. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Terlecky S. R., Nuttley W. M., McCollum D., Sock E., Subramani S. The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal. EMBO J. 1995 Aug 1;14(15):3627–3634. doi: 10.1002/j.1460-2075.1995.tb00032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tsukamoto T., Hata S., Yokota S., Miura S., Fujiki Y., Hijikata M., Miyazawa S., Hashimoto T., Osumi T. Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor. J Biol Chem. 1994 Feb 25;269(8):6001–6010. [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Veenhuis M., Mateblowski M., Kunau W. H., Harder W. Proliferation of microbodies in Saccharomyces cerevisiae. Yeast. 1987 Jun;3(2):77–84. doi: 10.1002/yea.320030204. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Watanabe M., Blobel G. High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10133–10136. doi: 10.1073/pnas.92.22.10133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Wiebel F. F., Kunau W. H. The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes. Nature. 1992 Sep 3;359(6390):73–76. doi: 10.1038/359073a0. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Yaffe M. P., Schatz G. Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4819–4823. doi: 10.1073/pnas.81.15.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. de Hoop M. J., Ab G. Import of proteins into peroxisomes and other microbodies. Biochem J. 1992 Sep 15;286(Pt 3):657–669. doi: 10.1042/bj2860657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. van der Klei I. J., Faber K. N., Keizer-Gunnink I., Gietl C., Harder W., Veenhuis M. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha. FEBS Lett. 1993 Nov 8;334(1):128–132. doi: 10.1016/0014-5793(93)81697-x. [DOI] [PubMed] [Google Scholar]
  63. van der Klei I. J., Hilbrands R. E., Swaving G. J., Waterham H. R., Vrieling E. G., Titorenko V. I., Cregg J. M., Harder W., Veenhuis M. The Hansenula polymorpha PER3 gene is essential for the import of PTS1 proteins into the peroxisomal matrix. J Biol Chem. 1995 Jul 21;270(29):17229–17236. doi: 10.1074/jbc.270.29.17229. [DOI] [PubMed] [Google Scholar]
  64. van der Voorn L., Ploegh H. L. The WD-40 repeat. FEBS Lett. 1992 Jul 28;307(2):131–134. doi: 10.1016/0014-5793(92)80751-2. [DOI] [PubMed] [Google Scholar]

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