Protein Targeting Protocols pp 373-391

Part of the Methods in Molecular Biology™ book series (MIMB, volume 390) | Cite as

Protein Targeting to Yeast Peroxisomes

  • Ida van der Klei
  • Marten Veenhuis


Peroxisomes are important organelles of eukaryote cells. Although these structures are of relatively small size, they display an unprecedented functional versatility. The principles of their biogenesis and function are strongly conserved from very simple eukaryotes to humans. Peroxisome-borne proteins are synthesized in the cytosol and posttranslationally incorporated into the organelle.The protein-sorting signal for matrix proteins, peroxisomal targeting signal (PTS), and for membrane proteins (mPTS), are also conserved. Several genes involved in peroxisomal matrix protein import have been identified (PEX genes), but the details of the molecular mechanisms of this translocation process are still unclear. Here we describe procedures to study the subcellular location of peroxisomal matrix and membrane proteins in yeast and fungi. Emphasis is placed on protocols developed for the methylotrophic yeast Hansenula polymorpha, but very similar protocols can be applied for other yeast species and filamentous fungi. The described methods include cell fractionation procedures and subcellular localization studies using fluorescence microscopy and immunolabeling techniques.

Key Words

Peroxisome yeast Hansenula polymorpha cell fractionation fluorescence microscopy immunolabeling 


  1. 1.
    Purdue, P. E. and Lazarow, P. B. (2001) Peroxisome biogenesis. Annu. Rev. Cell Dev. Biol. 17, 701–752.CrossRefPubMedGoogle Scholar
  2. 2.
    Heiland, I. and Erdmann, R. (2005) Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins. FEBS J. 272, 2362–2372.CrossRefPubMedGoogle Scholar
  3. 3.
    Kunau, W. H. (2005) Peroxisome biogenesis: end of the debate. Curr. Biol. 15, R774–776.CrossRefPubMedGoogle Scholar
  4. 4.
    Brown, L. A. and Baker, A. (2003) Peroxisome biogenesis and the role of protein import. J. Cell Mol. Med. 7, 388–400.CrossRefPubMedGoogle Scholar
  5. 5.
    Baker, A. and Sparkes, I. A. (2005) Peroxisome protein import: some answers, more questions. Curr. Opin. Plant Biol. 8, 640–647.CrossRefPubMedGoogle Scholar
  6. 6.
    Ozimek, P., Veenhuis, M., and van der Klei, I. J. (2005) Alcohol oxidase:a complex peroxisomal, oligomeric flavoprotein. FEMS Yeast Res. 5, 975–983.CrossRefPubMedGoogle Scholar
  7. 7.
    Klein, A. T., van den Berg, M., Bottger, G., Tabak, H. F., and Distel, B.(2002) Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p. J. Biol. Chem. 277, 25011–25019.CrossRefPubMedGoogle Scholar
  8. 8.
    Schafer, A., Kerssen, D., Veenhuis, M., Kunau, W. H., and Schliebs, W.(2004) Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N–terminal half of the PTS1 receptor Pex5p. Mol. Cell Biol. 24, 8895–8906.CrossRefPubMedGoogle Scholar
  9. 9.
    Yang, X., Purdue, P. E., and Lazarow, P. B. (2001) Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p. Eur. J. Cell Biol. 80,26–38.CrossRefGoogle Scholar
  10. 10.
    Baerends, R. J. S., Faber, K. N., Kram, A. M., Kiel, J. A. K. W., van der Klei, I. J., and Veenhuis, M. (2000) A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha pex3p is involved in incorporation of the protein into the peroxisomal membrane. J. Biol. Chem. 275, 9986–9995.CrossRefPubMedGoogle Scholar
  11. 11.
    Schliebs, W. and Kunau, W. H. (2004) Peroxisome membrane biogenesis: the stage is set. Curr. Biol. 14, R397–R399CrossRefPubMedGoogle Scholar
  12. 12.
    Rottensteiner, H., Kramer, A., Lorenzen, S., Stein, K., Landgraf, C., Volkmer-Engert, R., and Erdmann, R. (2004) Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals. Mol. Biol. Cell 15, 3406–3417.CrossRefPubMedGoogle Scholar
  13. 13.
    Lambkin, G. R. and Rachubinski, R. A. (2001) Yarrowia lipolytica cells mutant for the peroxisomal peroxin Pex19p contain structures resembling wild-type peroxisomes. Mol. Biol. Cell 12, 3353–3364.PubMedGoogle Scholar
  14. 14.
    Otzen, M., Perband, U., Wang, D., et al. (2004). Hansenula polymorpha Pex19p is essential for the formation of functional peroxisomal membranes. J. Biol. Chem. 279, 19181–19190.CrossRefPubMedGoogle Scholar
  15. 15.
    Baker, A., Charlton, W., Johnson, B, et al. (2000) Biochemical and molecular approaches to understanding protein import into peroxisomes. Biochem. Soc. Trans. 28, 499–504.CrossRefPubMedGoogle Scholar
  16. 16.
    Gouveia, A. M., Guimaraes, C. P., Oliveira, M. E., Reguenga, C., Sa-Miranda, C., and Azevedo, J. E. (2003) Characterization of the peroxisomal cycling receptor Pex5p import pathway. Adv. Exp. Med. Biol. 544, 219–220.PubMedGoogle Scholar
  17. 17.
    Miyata, N. and Fujiki, Y. (2005) Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export. Mol. Cell Biol. 25, 10822–10832.CrossRefPubMedGoogle Scholar
  18. 18.
    Platta, H. W., Grunau, S., Rosenkranz, K., Girzalsky, W., and Erdmann, R. (2005) Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol. Nat. Cell Biol. 7, 817–822.CrossRefPubMedGoogle Scholar
  19. 19.
    Matsuzono, Y. and Fujiki, Y. (2006) In vitro transport of membrane proteins to peroxisomes by shuttling receptor Pex19p. J. Biol. Chem. 281, 36–42.CrossRefPubMedGoogle Scholar
  20. 20.
    van Dijken, J. P., Otto, R., and Harder, W. (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch. Microbiol. 111, 137–144.CrossRefPubMedGoogle Scholar
  21. 21.
    Monastyrska. I., van der Heide, M., Krikken, A. M., Kiel, J.A., van der Klei, I. J., and Veenhuis, M. (2005) Atg8 is essential for macropexophagy in Hansenula polymorpha. Traffic 6, 66–74.CrossRefPubMedGoogle Scholar
  22. 22.
    Otzen, M., Wang, D., Lunenborg, M. G., and van der Klei, I. J. (2005) Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2). J. Cell Sci. 118, 3409–3418.CrossRefPubMedGoogle Scholar
  23. 23.
    de Vries, B., Todde, V., Stevens, P., Salomons, F., van der Klei, I. J., and Veenhuis, M. (2006) Pex14p is not required for N-starvation induced microautophagy and in catalytic amounts for macropexophagy in Hansenula polymorpha. Autophagy 2,183–188.PubMedGoogle Scholar
  24. 24.
    LaemmLi, U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefPubMedGoogle Scholar
  25. 25.
    Kyhse-Andersen, J. ( 1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10, 203–209.CrossRefPubMedGoogle Scholar
  26. 26.
    Faber, K. N., Haan, G. J., Baerends, R. J., Kram, A. M., and Veenhuis, M. (2002) Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p. J. Biol. Chem. 277, 11026–11033.CrossRefPubMedGoogle Scholar
  27. 27.
    Otzen, M., Krikken, A. M., Ozimek, P., et al. (2006) In the yeast Hansenula polymorpha peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins. FEMS Yeast Res. 6, 1157–1166.CrossRefPubMedGoogle Scholar
  28. 28.
    Haan, G. J., Baerends, R. J. S., Krikken, A. M., Otzen, M., Veenhuis, M. and van der Klei, I. J. ( 2006) Re-assembly of peroxisomes in Hansenula polymorpha pex3 cells upon re-introduction of Pex3p involves the nuclear envelope. FEMS Yeast Res. 6, 186–194.CrossRefPubMedGoogle Scholar
  29. 29.
    Faber, K. N., Haima, P., Harder, W., Veenhuis, M., and AB, G. (1994). Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr. Genet. 25, 305–310.CrossRefPubMedGoogle Scholar
  30. 30.
    Faber, K. N., Swaving, G. J., Faber, F., AB, G., Harder, W., Veenhuis, M., and Haima, P. (1992). Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. J. Gen. Microbiol. 138, 2405–2416.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Ida van der Klei
    • 1
  • Marten Veenhuis
    • 1
  1. 1.Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenThe Netherlands

Personalised recommendations