Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A new self-assembled peroxisomal vesicle required for efficient resealing of the plasma membrane

Nature Cell Biology volume 2, pages 226–231 (2000)Cite this article

Abstract

The Woronin body is a membrane-bound organelle that has been observed in over 50 species of filamentous fungi. However, neither the composition nor the precise function of the Woronin body has yet been determined. Here we purify the Woronin body from Neurospora crassa and isolate Hex1, a new protein containing a consensus sequence known as peroxisome-targeting signal-1 (PTS1). We show that Hex1 is localized to the matrix of the Woronin body by immunoelectron microscopy, and that a green fluorescent protein– (GFP–)Hex1 fusion protein is targeted to yeast peroxisomes in a PTS1- and peroxin-dependent manner. The expression of the HEX1 gene in yeast generates hexagonal vesicles that are morphologically similar to the native Woronin body, implying a Hex1-encoded mechanism of Woronin-body assembly. Deletion of HEX1 in N. crassa eliminates Woronin bodies from the cytoplasm and results in hyphae that exhibit a cytoplasmic-bleeding phenotype in response to cell lysis. Our results show that the Woronin body represents a new category of peroxisome with a function in the maintenance of cellular integrity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Appearance and purification of Woronin body from N. crassa.
Figure 2: Alignment of proteins with significant homology to Hex1.
Figure 3: Characterization of Hex1 by western blotting and immunoelectron microscopy.
Figure 4: Peroxisomal targeting and assembly of Hex1 into hexagonal granules in vivo and in vitro.
Figure 5: Analysis of a HEX1-deletion strain.
Figure 6: Cytoplasmic bleeding in the HEX1-deletion strain.

Similar content being viewed by others

References

  1. Woronin, M. Zur Entwicklungsgeschichte der Ascobolus pulcherrimus Cr. und einiger Pezizen. Abh. Senkenb. Naturforsch. 5, 333–344 (1864).

    Google Scholar 

  2. Trinci, A. P. & Collinge, A. J. Occlusion of the septal pores of damaged hyphae of Neurospora crassa by hexagonal crystals. Protoplasma 80, 57–67 (1974).

    Article  CAS  Google Scholar 

  3. Collinge, A. J. & Markham, P. Woronin bodies rapidly plug septal pores of severed Penicillium chrysogenum hyphae. Exp. Mycol. 9, 80–85 (1985).

    Article  Google Scholar 

  4. Markham, P. & Collinge, A. J. Woronin bodies in filamentous fungi. FEMS Microbiol. Rev. 46, 1–11 (1987).

    Article  Google Scholar 

  5. Hoch, H. C. & Maxwell, D. P. Proteinaceous hexagonal inclusions in hyphae of Whetzelinia sclerotiorum and Neurospora crassa. Can. J. Microbiol. 20, 1029–1035 (1974).

    Article  CAS  Google Scholar 

  6. McKeen, W. E. Woronin bodies in Erysiphe graminis DC. Can. J. Microbiol. 17, 1557–1563 (1971).

    Article  CAS  Google Scholar 

  7. Wergin, W. P. Development of Woronin bodies from microbodies in Fusarium oxysporum f. sp. lycopersici. Protoplasma 76, 249-260 (1973).

    Article  Google Scholar 

  8. Garrison, R. G., Lane, J. W. & Field, M. F. Ultrastructural changes during the yeastlike to mycelial-phase conversion of Blastomyces dermatitidis and Histoplasma capsalatum. J. Bacteriol. 101, 628–635 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Keller, G. A. et al. Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J. Cell Biol. 114, 893–904 (1991).

    Article  CAS  Google Scholar 

  10. 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. USA 73, 2043–2046 (1976).

    Article  CAS  Google Scholar 

  11. Subramani, S. Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement. Physiol. Rev. 78, 171–188 (1998).

    Article  CAS  Google Scholar 

  12. Olsen, L. J. The surprising complexity of peroxisome biogenesis. Plant Mol. Biol. 38, 163–189 (1998).

    Article  CAS  Google Scholar 

  13. Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J. & Subramani, S. A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol. 108, 1657–1664 (1989).

    Article  CAS  Google Scholar 

  14. Monosov, E. Z., Wenzel, T. J., Luers, G. H., Heyman, J. A. & Subramani, S. Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells. J. Histochem. Cytochem. 44, 581–589 (1996).

    Article  CAS  Google Scholar 

  15. 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. 10, 3255–3262 (1991).

    Article  CAS  Google Scholar 

  16. Tabak, H. F., Braakman, I. & Distel, B. Peroxisomes: simple in function but complex in maintenance. Trends Cell Biol. 9, 447–453 (1999).

    Article  CAS  Google Scholar 

  17. Nishimura, M., Hayashi, M., Kato, A., Yamaguchi, K. & Mano, S. Functional transformation of microbodies in higher plant cells. Cell Struct. Funct. 21, 387–393 (1996).

    Article  CAS  Google Scholar 

  18. Zuk, D. & Jacobson, A. A single amino acid substitution in yeast eIF-5A results in mRNA stabilization. EMBO J. 17, 2914–2925 (1998).

    Article  CAS  Google Scholar 

  19. Schatz, O. et al. Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5. Proc. Natl Acad. Sci. USA 95, 1607–1612 (1998).

    Article  CAS  Google Scholar 

  20. Park, M. H., Wolff, E. C. & Folk, J. E. Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. Biofactors 4, 95–104 (1993).

    CAS  PubMed  Google Scholar 

  21. Erdmann, R. & Blobel, G. Identification of Pex13p a peroxisomal membrane receptor for the PTS 1 recognition factor. J. Cell Biol. 135, 111–121 (1996).

    Article  CAS  Google Scholar 

  22. Wanner, G. & Theimer, R. R. Two types of microbodies in Neurospora crassa. Ann. NY Acad. Sci. 386, 269–284 (1982).

    Article  CAS  Google Scholar 

  23. de Zoysa, P. A. & Connerton, I. F. The function and specificity of the C-terminal tripeptide glyoxysomal targeting signal in Neurospora crassa. Curr. Genet. 26, 430–437 (1994).

    Article  CAS  Google Scholar 

  24. Kionka, C. & Kunau, W. H. Inducible beta-oxidation pathway in Neurospora crassa. J. Bacteriol. 161, 153–157 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Steinhardt, R. A., Bi, G. & Alderton, J. M. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263, 390–393 (1994).

    Article  CAS  Google Scholar 

  26. Bi, G. Q. et al. Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis. J. Cell Biol. 138, 999–1008 (1997).

    Article  CAS  Google Scholar 

  27. Vogel, H. J. Distribution of lysine pathways among fungi: evolutionary implications. Am. Natur. 98, 435–446 (1964).

    Article  CAS  Google Scholar 

  28. Rose, M., Winston, F. & Heiter, P. Methods in Yeast Genetics (Cold Spring Harb. Lab. Press, Cold Spring Harbor, 1990).

  29. Guan, K. L. & Dixon, J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192, 262–267 (1991).

    Article  CAS  Google Scholar 

  30. Mumberg, D., Muller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995).

    Article  CAS  Google Scholar 

  31. Oancea, E. & Meyer, T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307–318 (1998).

    Article  CAS  Google Scholar 

  32. Staben, C. et al. Use of a bacterial Hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet. Newsletter 36, 79–81 (1989).

    Google Scholar 

  33. Margolin, B. S., Freitag, M. & Selker, E. U. Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Genet. Newsletter 44, 34–36 (1997).

    Google Scholar 

  34. Jedd, G., Mulholland, J. & Segev, N. Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment. J. Cell Biol. 137, 563–580 (1997).

    Article  CAS  Google Scholar 

  35. Pringle, J. R. et al. Fluorescence microscopy methods for yeast. Methods Cell Biol. 31, 357–435 (1989).

    Article  CAS  Google Scholar 

  36. Byers, B. & Boetsch, L. Preparation of yeast cells for thin-section electron microscopy. Methods Enzymol. 194, 602–608 (1991).

    Article  CAS  Google Scholar 

  37. Erdmann, R. & Blobel, G. Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J. Cell Biol. 128, 509–523 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Shio for help with electron microscopy, J. Fernandez for protein sequencing, A. Jacobsen for the eIF-5 A mutant and F. Bartolini for comments on the manuscript. G.J. is supported by a postdoctoral fellowship from the NIH, no. GM 19828-02. This work was supported in part by NSF grant no. 9420038 to N-H.C.

Correspondence and requests for materials should be addressed to N-H.C.

Sequences used in the alignment presented in Fig. 2 have been deposited in GenBank under the following accession numbers: Neurospora crassa HEX1, AF001033; Aspergillus nidulans HEX1, AF23965; Magnaporthe grisea HEX1, AF170544; Botrytis cinerea HEX1, AL116826; Saccharomyces cerevisiae EIF-5 A, M63542.

Author information

Authors and Affiliations

  1. Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, 10021-6399, New York, USA

    Gregory Jedd & Nam-Hai Chua

Authors
  1. Gregory Jedd
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nam-Hai Chua
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nam-Hai Chua.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jedd, G., Chua, NH. A new self-assembled peroxisomal vesicle required for efficient resealing of the plasma membrane. Nat Cell Biol 2, 226–231 (2000). https://doi.org/10.1038/35008652

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35008652

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing