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:

Crystal structure of peroxisomal targeting signal-2 bound to its receptor complex Pex7p–Pex21p

Nature Structural & Molecular Biology volume 20, pages 987–993 (2013)Cite this article

Subjects

Abstract

Appropriate targeting of matrix proteins to peroxisomes is mainly directed by two types of peroxisomal targeting signals, PTS1 and PTS2. Although the basis of PTS1 recognition has been revealed by structural studies, that of PTS2 recognition remains elusive. Here we present the crystal structure of a heterotrimeric PTS2-recognition complex from Saccharomyces cerevisiae, containing Pex7p, the C-terminal region of Pex21p and the PTS2 of the peroxisomal 3-ketoacyl-CoA thiolase. Pex7p forms a β-propeller structure and provides a platform for cooperative interactions with both the amphipathic PTS2 helix and Pex21p. The C-terminal region of Pex21p directly covers the hydrophobic surfaces of both Pex7p and PTS2, and the resulting hydrophobic core is the primary determinant of stable complex formation. Together with in vivo and in vitro functional assays of Pex7p and Pex21p variants, our findings reveal the molecular mechanism of PTS2 recognition.

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: Structure of the Pex7p–Pex21pC–Fox3pN-MBP complex.
Figure 2: Cooperative complex formation of Fox3p, Pex7p and Pex21pC.
Figure 3: Pex7p–Pex21pC receptor complex formation.
Figure 4: Molecular recognition of key residues of PTS2.
Figure 5: Functional assays of Pex7p and Pex21p variants.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Wanders, R.J. & Waterham, H.R. Biochemistry of mammalian peroxisomes revisited. Annu. Rev. Biochem. 75, 295–332 (2006).

    Article  CAS  Google Scholar 

  2. Platta, H.W. & Erdmann, R. Peroxisomal dynamics. Trends Cell Biol. 17, 474–484 (2007).

    Article  CAS  Google Scholar 

  3. Ma, C., Agrawal, G. & Subramani, S. Peroxisome assembly: matrix and membrane protein biogenesis. J. Cell Biol. 193, 7–16 (2011).

    Article  CAS  Google Scholar 

  4. Steinberg, S.J. et al. Peroxisome biogenesis disorders. Biochim. Biophys. Acta 1763, 1733–1748 (2006).

    Article  CAS  Google Scholar 

  5. Stanley, W.A. et al. Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p. Mol. Cell 24, 653–663 (2006).

    Article  CAS  Google Scholar 

  6. Gatto, G.J. Jr., Geisbrecht, B.V., Gould, S.J. & Berg, J.M. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat. Struct. Biol. 7, 1091–1095 (2000).

    Article  CAS  Google Scholar 

  7. Lazarow, P.B. The import receptor Pex7p and the PTS2 targeting sequence. Biochim. Biophys. Acta 1763, 1599–1604 (2006).

    Article  CAS  Google Scholar 

  8. Schliebs, W. & Kunau, W.H. PTS2 co-receptors: diverse proteins with common features. Biochim. Biophys. Acta 1763, 1605–1612 (2006).

    Article  CAS  Google Scholar 

  9. 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 

  10. Osumi, T. et al. Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem. Biophys. Res. Commun. 181, 947–954 (1991).

    Article  CAS  Google Scholar 

  11. Petriv, O.I., Tang, L., Titorenko, V.I. & Rachubinski, R.A. A new definition for the consensus sequence of the peroxisome targeting signal type 2. J. Mol. Biol. 341, 119–134 (2004).

    Article  CAS  Google Scholar 

  12. 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. 13, 4908–4918 (1994).

    Article  CAS  Google Scholar 

  13. 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. 129, 65–80 (1995).

    Article  CAS  Google Scholar 

  14. Braverman, N. et al. Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata. Nat. Genet. 15, 369–376 (1997).

    Article  CAS  Google Scholar 

  15. Motley, A.M. et al. Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor. Nat. Genet. 15, 377–380 (1997).

    Article  CAS  Google Scholar 

  16. 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. 15, 381–384 (1997).

    Article  CAS  Google Scholar 

  17. Elgersma, Y. et al. A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris. J. Cell Biol. 140, 807–820 (1998).

    Article  CAS  Google Scholar 

  18. Mukai, S., Ghaedi, K. & Fujiki, Y. Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells. J. Biol. Chem. 277, 9548–9561 (2002).

    Article  CAS  Google Scholar 

  19. Nito, K., Hayashi, M. & Nishimura, M. Direct interaction and determination of binding domains among peroxisomal import factors in Arabidopsis thaliana. Plant Cell Physiol. 43, 355–366 (2002).

    Article  CAS  Google Scholar 

  20. Sichting, M., Schell-Steven, A., Prokisch, H., Erdmann, R. & Rottensteiner, H. Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes. Mol. Biol. Cell 14, 810–821 (2003).

    Article  CAS  Google Scholar 

  21. Rehling, P. et al. The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene. EMBO J. 15, 2901–2913 (1996).

    Article  CAS  Google Scholar 

  22. 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. 269, 7558–7563 (1994).

    CAS  PubMed  Google Scholar 

  23. Braverman, N. et al. Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype. Hum. Mutat. 20, 284–297 (2002).

    Article  CAS  Google Scholar 

  24. Motley, A.M. et al. Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am. J. Hum. Genet. 70, 612–624 (2002).

    Article  CAS  Google Scholar 

  25. Kunze, M. et al. Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7. J. Biol. Chem. 286, 45048–45062 (2011).

    Article  CAS  Google Scholar 

  26. Purdue, P.E., Yang, X. & Lazarow, P.B. Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J. Cell Biol. 143, 1859–1869 (1998).

    Article  CAS  Google Scholar 

  27. Einwächter, H., Sowinski, S., Kunau, W.H. & Schliebs, W. Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway. EMBO Rep. 2, 1035–1039 (2001).

    Article  Google Scholar 

  28. Léon, S. et al. Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation. J. Cell Biol. 172, 67–78 (2006).

    Article  Google Scholar 

  29. Khan, B.R. & Zolman, B.K. Pex5 mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis. Plant Physiol. 154, 1602–1615 (2010).

    Article  CAS  Google Scholar 

  30. 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. 7, 1195–1205 (1998).

    Article  CAS  Google Scholar 

  31. Otera, H. et al. 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. 18, 388–399 (1998).

    Article  CAS  Google Scholar 

  32. Matsumura, T., Otera, H. & Fujiki, Y. Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals: study with a novel Pex5-impaired Chinese hamster ovary cell mutant. J. Biol. Chem. 275, 21715–21721 (2000).

    Article  CAS  Google Scholar 

  33. Dodt, G., Warren, D., Becker, E., Rehling, P. & Gould, S.J. Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J. Biol. Chem. 276, 41769–41781 (2001).

    Article  CAS  Google Scholar 

  34. Léon, S. & Subramani, S. A conserved cysteine residue of Pichia pastoris Pex20p is essential for its recycling from the peroxisome to the cytosol. J. Biol. Chem. 282, 7424–7430 (2007).

    Article  Google Scholar 

  35. Hensel, A. et al. Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane. J. Biol. Chem. 286, 43495–43505 (2011).

    Article  CAS  Google Scholar 

  36. Neufeld, C. et al. Structural basis for competitive interactions of Pex14 with the import receptors Pex5 and Pex19. EMBO J. 28, 745–754 (2009).

    Article  CAS  Google Scholar 

  37. Smith, T.F., Gaitatzes, C., Saxena, K. & Neer, E.J. The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci. 24, 181–185 (1999).

    Article  CAS  Google Scholar 

  38. Wall, M.A. et al. The structure of the G protein heterotrimer Giα1β1γ2 . Cell 83, 1047–1058 (1995).

    Article  CAS  Google Scholar 

  39. Couture, J.F., Collazo, E. & Trievel, R.C. Molecular recognition of histone H3 by the WD40 protein WDR5. Nat. Struct. Mol. Biol. 13, 698–703 (2006).

    Article  CAS  Google Scholar 

  40. Janda, C.Y. et al. Recognition of a signal peptide by the signal recognition particle. Nature 465, 507–510 (2010).

    Article  CAS  Google Scholar 

  41. Abe, Y. et al. Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100, 551–560 (2000).

    Article  CAS  Google Scholar 

  42. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    Article  CAS  Google Scholar 

  43. Wu, S. & Letchworth, G.J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152–154 (2004).

    Article  CAS  Google Scholar 

  44. Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  Google Scholar 

  45. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  Google Scholar 

  46. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  47. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  49. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  50. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  51. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  52. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. & Cullin, C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329–3330 (1993).

    Article  CAS  Google Scholar 

  53. Gietz, R.D. & Schiestl, R.H. Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 1–4 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Hirai for helpful advice on plasmid construction, protein purification and crystallization. We thank Y. Matsuura for critical reading of the manuscript. This work was supported by the Targeted Proteins Research Program of the Ministry of Education, Culture, Sports, Science, and Technology, Japan (H.K.). The synchrotron radiation experiments were performed at SPring-8 BL41XU (2011A1210, 2011A1289).

Author information

Authors and Affiliations

  1. Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Dongqing Pan, Toru Nakatsu & Hiroaki Kato

  2. RIKEN SPring-8 Center, Sayo, Japan

    Toru Nakatsu & Hiroaki Kato

Authors
  1. Dongqing Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toru Nakatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroaki Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.P. generated plasmids, proteins and crystals. D.P. and T.N. collected diffraction data and solved the structure. D.P. performed pulldown experiments and in vivo functional assays. H.K. supervised the project. D.P. and H.K. wrote the paper.

Corresponding authors

Correspondence to Dongqing Pan or Hiroaki Kato.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Note (PDF 1429 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pan, D., Nakatsu, T. & Kato, H. Crystal structure of peroxisomal targeting signal-2 bound to its receptor complex Pex7p–Pex21p. Nat Struct Mol Biol 20, 987–993 (2013). https://doi.org/10.1038/nsmb.2618

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2618

This article is cited by

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