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Biochemical Journal logoLink to Biochemical Journal
. 2000 Feb 15;346(Pt 1):177–184.

Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p.

R K Szilard 1, R A Rachubinski 1
PMCID: PMC1220838  PMID: 10657255

Abstract

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.

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Selected References

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  1. Aitchison J. D., Murray W. W., Rachubinski R. A. The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes. J Biol Chem. 1991 Dec 5;266(34):23197–23203. [PubMed] [Google Scholar]
  2. Aitchison J. D., Szilard R. K., Nuttley W. M., Rachubinski R. A. Antibodies directed against a yeast carboxyl-terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts. Yeast. 1992 Sep;8(9):721–734. doi: 10.1002/yea.320080905. [DOI] [PubMed] [Google Scholar]
  3. Albertini M., Rehling P., Erdmann R., Girzalsky W., Kiel J. A., Veenhuis M., Kunau W. H. Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent import pathways. Cell. 1997 Apr 4;89(1):83–92. doi: 10.1016/s0092-8674(00)80185-3. [DOI] [PubMed] [Google Scholar]
  4. Baes M., Gressens P., Baumgart E., Carmeliet P., Casteels M., Fransen M., Evrard P., Fahimi D., Declercq P. E., Collen D. A mouse model for Zellweger syndrome. Nat Genet. 1997 Sep;17(1):49–57. doi: 10.1038/ng0997-49. [DOI] [PubMed] [Google Scholar]
  5. Braverman N., Dodt G., Gould S. J., Valle D. An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum Mol Genet. 1998 Aug;7(8):1195–1205. doi: 10.1093/hmg/7.8.1195. [DOI] [PubMed] [Google Scholar]
  6. Braverman N., Steel G., Obie C., Moser A., Moser H., Gould S. J., Valle D. Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata. Nat Genet. 1997 Apr;15(4):369–376. doi: 10.1038/ng0497-369. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Brocard C., Lametschwandtner G., Koudelka R., Hartig A. Pex14p is a member of the protein linkage map of Pex5p. EMBO J. 1997 Sep 15;16(18):5491–5500. doi: 10.1093/emboj/16.18.5491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Elgersma Y., Elgersma-Hooisma M., Wenzel T., McCaffery J. M., Farquhar M. G., Subramani S. A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris. J Cell Biol. 1998 Feb 23;140(4):807–820. doi: 10.1083/jcb.140.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elgersma Y., Kwast L., Klein A., Voorn-Brouwer T., van den Berg M., Metzig B., America T., Tabak H. F., Distel B. The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins. J Cell Biol. 1996 Oct;135(1):97–109. doi: 10.1083/jcb.135.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Erdmann R., Blobel G. Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor. J Cell Biol. 1996 Oct;135(1):111–121. doi: 10.1083/jcb.135.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Fransen M., Terlecky S. R., Subramani S. Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8087–8092. doi: 10.1073/pnas.95.14.8087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Girzalsky W., Rehling P., Stein K., Kipper J., Blank L., Kunau W. H., Erdmann R. Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes. J Cell Biol. 1999 Mar 22;144(6):1151–1162. doi: 10.1083/jcb.144.6.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Gould S. J., Kalish J. E., Morrell J. C., Bjorkman J., Urquhart A. J., Crane D. I. Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor. J Cell Biol. 1996 Oct;135(1):85–95. doi: 10.1083/jcb.135.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hettema E. H., Distel B., Tabak H. F. Import of proteins into peroxisomes. Biochim Biophys Acta. 1999 Aug 12;1451(1):17–34. doi: 10.1016/s0167-4889(99)00087-7. [DOI] [PubMed] [Google Scholar]
  22. Huhse B., Rehling P., Albertini M., Blank L., Meller K., Kunau W. H. Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the peroxisomal protein translocation machinery. J Cell Biol. 1998 Jan 12;140(1):49–60. doi: 10.1083/jcb.140.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Komori M., Rasmussen S. W., Kiel J. A., Baerends R. J., Cregg J. M., van der Klei I. J., Veenhuis M. The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis. EMBO J. 1997 Jan 2;16(1):44–53. doi: 10.1093/emboj/16.1.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  25. Kragler F., Lametschwandtner G., Christmann J., Hartig A., Harada J. J. Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13336–13341. doi: 10.1073/pnas.95.22.13336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Motley A. M., Hettema E. H., Hogenhout E. M., Brites P., ten Asbroek A. L., Wijburg F. A., Baas F., Heijmans H. S., Tabak H. F., Wanders R. J. Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor. Nat Genet. 1997 Apr;15(4):377–380. doi: 10.1038/ng0497-377. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Otera H., Okumoto K., Tateishi K., Ikoma Y., Matsuda E., Nishimura M., Tsukamoto T., Osumi T., Ohashi K., Higuchi O. Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants. Mol Cell Biol. 1998 Jan;18(1):388–399. doi: 10.1128/mcb.18.1.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Purdue P. E., Zhang J. W., Skoneczny M., Lazarow P. B. Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor. Nat Genet. 1997 Apr;15(4):381–384. doi: 10.1038/ng0497-381. [DOI] [PubMed] [Google Scholar]
  34. Rachubinski R. A., Subramani S. How proteins penetrate peroxisomes. Cell. 1995 Nov 17;83(4):525–528. doi: 10.1016/0092-8674(95)90091-8. [DOI] [PubMed] [Google Scholar]
  35. Rehling P., Marzioch M., Niesen F., Wittke E., Veenhuis M., Kunau W. H. The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene. EMBO J. 1996 Jun 17;15(12):2901–2913. [PMC free article] [PubMed] [Google Scholar]
  36. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  37. Schliebs W., Saidowsky J., Agianian B., Dodt G., Herberg F. W., Kunau W. H. Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14. J Biol Chem. 1999 Feb 26;274(9):5666–5673. doi: 10.1074/jbc.274.9.5666. [DOI] [PubMed] [Google Scholar]
  38. Shimizu N., Itoh R., Hirono Y., Otera H., Ghaedi K., Tateishi K., Tamura S., Okumoto K., Harano T., Mukai S. The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis. J Biol Chem. 1999 Apr 30;274(18):12593–12604. doi: 10.1074/jbc.274.18.12593. [DOI] [PubMed] [Google Scholar]
  39. Subramani S. Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement. Physiol Rev. 1998 Jan;78(1):171–188. doi: 10.1152/physrev.1998.78.1.171. [DOI] [PubMed] [Google Scholar]
  40. Szilard R. K., Titorenko V. I., Veenhuis M., Rachubinski R. A. Pay32p of the yeast Yarrowia lipolytica is an intraperoxisomal component of the matrix protein translocation machinery. J Cell Biol. 1995 Dec;131(6 Pt 1):1453–1469. doi: 10.1083/jcb.131.6.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. The electronic plant gene register. Plant Physiol. 1999 May;120(1):339–340. doi: 10.1104/pp.120.1.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Will G. K., Soukupova M., Hong X., Erdmann K. S., Kiel J. A., Dodt G., Kunau W. H., Erdmann R. Identification and characterization of the human orthologue of yeast Pex14p. Mol Cell Biol. 1999 Mar;19(3):2265–2277. doi: 10.1128/mcb.19.3.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wimmer C., Schmid M., Veenhuis M., Gietl C. The plant PTS1 receptor: similarities and differences to its human and yeast counterparts. Plant J. 1998 Nov;16(4):453–464. doi: 10.1046/j.1365-313x.1998.00320.x. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Zhang J. W., Lazarow P. B. 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. J Cell Biol. 1996 Feb;132(3):325–334. doi: 10.1083/jcb.132.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]

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