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. 1995 Dec 2;131(6):1453–1469. doi: 10.1083/jcb.131.6.1453

Pay32p of the yeast Yarrowia lipolytica is an intraperoxisomal component of the matrix protein translocation machinery

PMCID: PMC2120665  PMID: 8522603

Abstract

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay32-1, has abnormally small peroxisomes that are often found in clusters surrounded by membraneous material. The functionally complementing gene PAY32 encodes a protein, Pay32p, of 598 amino acids (66,733 D) that is a member of the tetratricopeptide repeat family. Pay32p is intraperoxisomal. In wild-type peroxisomes, Pay32p is associated primarily with the inner surface of the peroxisomal membrane, but approximately 30% of Pay32p is localized to the peroxisomal matrix. The majority of Pay32p in the matrix is complexed with two polypeptides of 62 and 64 kD recognized by antibodies to SKL (peroxisomal targeting signal-1). In contrast, in peroxisomes of the pay32-1 mutant, Pay32p is localized exclusively to the matrix and forms no complex. Biochemical characterization of the mutants pay32-1 and pay32-KO (a PAY32 gene disruption strain) showed that Pay32p is a component of the peroxisomal translocation machinery. Mutations in the PAY32 gene prevent the translocation of most peroxisome-bound proteins into the peroxisomal matrix. These proteins, including the 62-kD anti-SKL-reactive polypeptide, are trapped in the peroxisomal membrane at an intermediate stage of translocation in pay32 mutants. Our results suggest that there are at least two distinct translocation machineries involved in the import of proteins into peroxisomes.

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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. Barth G., Weber H. Genetic analysis of the gene ICL1 of the yeast Yarrowia lipolytica. Yeast. 1987 Dec;3(4):255–262. doi: 10.1002/yea.320030406. [DOI] [PubMed] [Google Scholar]
  4. Bellion E., Goodman J. M. Proton ionophores prevent assembly of a peroxisomal protein. Cell. 1987 Jan 16;48(1):165–173. doi: 10.1016/0092-8674(87)90367-9. [DOI] [PubMed] [Google Scholar]
  5. Berninger G., Schmidtchen R., Casel G., Knörr A., Rautenstrauss K., Kunau W. H., Schweizer E. Structure and metabolic control of the Yarrowia lipolytica peroxisomal 3-oxoacyl-CoA-thiolase gene. Eur J Biochem. 1993 Sep 1;216(2):607–613. doi: 10.1111/j.1432-1033.1993.tb18180.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. De Duve C., Baudhuin P. Peroxisomes (microbodies and related particles). Physiol Rev. 1966 Apr;46(2):323–357. doi: 10.1152/physrev.1966.46.2.323. [DOI] [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. Eitzen G. A., Aitchison J. D., Szilard R. K., Veenhuis M., Nuttley W. M., Rachubinski R. A. The Yarrowia lipolytica gene PAY2 encodes a 42-kDa peroxisomal integral membrane protein essential for matrix protein import and peroxisome enlargement but not for peroxisome membrane proliferation. J Biol Chem. 1995 Jan 20;270(3):1429–1436. doi: 10.1074/jbc.270.3.1429. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Franzusoff A., Rothblatt J., Schekman R. Analysis of polypeptide transit through yeast secretory pathway. Methods Enzymol. 1991;194:662–674. doi: 10.1016/0076-6879(91)94048-h. [DOI] [PubMed] [Google Scholar]
  14. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gaillardin C. M., Charoy V., Heslot H. A study of copulation, sporulation and meiotic segregation in Candida lipolytica. Arch Mikrobiol. 1973;92(1):69–83. doi: 10.1007/BF00409513. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Goodman J. M., Trapp S. B., Hwang H., Veenhuis M. Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins. J Cell Sci. 1990 Sep;97(Pt 1):193–204. doi: 10.1242/jcs.97.1.193. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Gratzer S., Lithgow T., Bauer R. E., Lamping E., Paltauf F., Kohlwein S. D., Haucke V., Junne T., Schatz G., Horst M. Mas37p, a novel receptor subunit for protein import into mitochondria. J Cell Biol. 1995 Apr;129(1):25–34. doi: 10.1083/jcb.129.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heyman J. A., Monosov E., Subramani S. Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis. J Cell Biol. 1994 Dec;127(5):1259–1273. doi: 10.1083/jcb.127.5.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Lazarow P. B., De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2043–2046. doi: 10.1073/pnas.73.6.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Lithgow T., Glick B. S., Schatz G. The protein import receptor of mitochondria. Trends Biochem Sci. 1995 Mar;20(3):98–101. doi: 10.1016/s0968-0004(00)88972-0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  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. Miura S., Kasuya-Arai I., Mori H., Miyazawa S., Osumi T., Hashimoto T., Fujiki Y. Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal. J Biol Chem. 1992 Jul 15;267(20):14405–14411. [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. Nuttley W. M., Bodnar A. G., Mangroo D., Rachubinski R. A. Isolation and characterization of membranes from oleic acid-induced peroxisomes of Candida tropicalis. J Cell Sci. 1990 Mar;95(Pt 3):463–470. doi: 10.1242/jcs.95.3.463. [DOI] [PubMed] [Google Scholar]
  40. Nuttley W. M., Brade A. M., Eitzen G. A., Veenhuis M., Aitchison J. D., Szilard R. K., Glover J. R., Rachubinski R. A. PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases. J Biol Chem. 1994 Jan 7;269(1):556–566. [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. Osumi T., Hashimoto T. Peroxisomal beta oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. Biochem Biophys Res Commun. 1979 Jul 27;89(2):580–584. doi: 10.1016/0006-291x(79)90669-7. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Purdue P. E., Takada Y., Danpure C. J. Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1. J Cell Biol. 1990 Dec;111(6 Pt 1):2341–2351. doi: 10.1083/jcb.111.6.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Roggenkamp R. Targeting signals for protein import into peroxisomes. Cell Biochem Funct. 1992 Sep;10(3):193–199. doi: 10.1002/cbf.290100309. [DOI] [PubMed] [Google Scholar]
  49. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  50. Sikorski R. S., Michaud W. A., Hieter P. p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains. Mol Cell Biol. 1993 Feb;13(2):1212–1221. doi: 10.1128/mcb.13.2.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. 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]
  54. 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]
  55. Swinkels B. W., Gould S. J., Subramani S. Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett. 1992 Jun 29;305(2):133–136. doi: 10.1016/0014-5793(92)80880-p. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. 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]
  59. Voos W., Gambill B. D., Laloraya S., Ang D., Craig E. A., Pfanner N. Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes. Mol Cell Biol. 1994 Oct;14(10):6627–6634. doi: 10.1128/mcb.14.10.6627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Walton P. A., Gould S. J., Rachubinski R. A., Subramani S., Feramisco J. R. Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal. J Cell Biol. 1992 Aug;118(3):499–508. doi: 10.1083/jcb.118.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Walton P. A., Wendland M., Subramani S., Rachubinski R. A., Welch W. J. Involvement of 70-kD heat-shock proteins in peroxisomal import. J Cell Biol. 1994 Jun;125(5):1037–1046. doi: 10.1083/jcb.125.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Waterham H. R., Titorenko V. I., Swaving G. J., Harder W., Veenhuis M. Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles. EMBO J. 1993 Dec;12(12):4785–4794. doi: 10.1002/j.1460-2075.1993.tb06167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]
  64. 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]
  65. 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]
  66. 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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