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.

  • Letter
  • Published:

Targeting of proteins to membranes through hedgehog auto-processing

Nature Biotechnology volume 21, pages 936–940 (2003)Cite this article

A Corrigendum to this article was published on 01 September 2003

Abstract

Hedgehog proteins use an auto-processing strategy to generate cholesterol-conjugated peptide products that act as extracellular ligands in a number of developmental signaling pathways. We describe an approach that takes advantage of the hedgehog auto-processing reaction to carry out intracellular modification of heterologous proteins, resulting in their localization to cell membranes. Such processing occurs spontaneously, without accessory proteins or modification by other enzymes. Using the green fluorescent protein (GFP) and the product of the Hras as model proteins, we demonstrate the use of hedgehog auto-processing to process heterologous N-terminal domains and direct the resulting biologically active products to cell membranes. This system represents a tool for targeting functional peptides and proteins to cell membranes, and may also offer a means of directing peptides or other small molecules to components of cholesterol metabolism or regulation.

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: Expression and characterization of GFP fused to the hedgehog C-terminal autoprocessing domain.
Figure 2: Analysis of hedgehog-processed GFP proteins.
Figure 3: Transformation activity of hedgehog-processed V12Ras.

Similar content being viewed by others

References

  1. Clarke, S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu. Rev. Biochem. 61, 355–386 (1992).

    Article  CAS  Google Scholar 

  2. Rogers, W. Making membranes green: construction and characterization of GFP-fusion proteins targeted to discrete plasma membrane domains. Biotechniques 5, 1044–1046 (2002).

    Article  Google Scholar 

  3. Zacharias, D., Violin, J., Newton, A. & Tsien, R. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    Article  CAS  Google Scholar 

  4. Porter, J.A., Young, K.E. & Beachy, P.A. Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255–259 (1996).

    Article  CAS  Google Scholar 

  5. Porter, J.A. et al. Hedgehog patterning activity: Role of lipophilic modification mediated by the carboxyl-terminal autoprocessing domain. Cell 86, 21–34 (1996).

    Article  CAS  Google Scholar 

  6. Ingham, P.W. Hedgehog signaling: a tale of two lipids. Science 294, 1879–1881 (2001).

    Article  CAS  Google Scholar 

  7. Casey, P.J., Solski, P.A., Der, C.J. & Buss, J.E. p21ras is modified by a farnesyl isoprenoid. Proc. Natl. Acad. Sci. USA 86, 8323–8627 (1989).

    Article  CAS  Google Scholar 

  8. Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607 (1981).

    CAS  PubMed  Google Scholar 

  9. Hall, T.M. et al. Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins. Cell 91, 85–97 (1997).

    Article  CAS  Google Scholar 

  10. Smart, E.J. et al. Caveolins, liquid-ordered domains, and signal transduction. Mol. Cell. Biol. 19, 7289–7304 (1999).

    Article  CAS  Google Scholar 

  11. Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell. Biol. 1, 31–39 (2000).

    Article  CAS  Google Scholar 

  12. Simons, K. & Ikonen, E. How cells handle cholesterol. Science 290, 1721–1726 (2000).

    Article  CAS  Google Scholar 

  13. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  CAS  Google Scholar 

  14. Simons, K. & Ehehalt, R. Cholesterol, lipid rafts and diseases. J. Clin. Invest. 110, 597–603 (2002).

    Article  CAS  Google Scholar 

  15. Song, K.S. et al. Co-purification and direct interaction of Ras with Caveolin, an integral membrane protein of caveolae microdomains. J. Biol. Chem. 271, 9690–9697 (1986).

    Article  Google Scholar 

  16. Albritton, L.M., Tseng, L., Scadden, D. & Cunningham, J.M. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57, 659–666 (1989).

    Article  CAS  Google Scholar 

  17. Covic, L., Gresser, A.L., Talavera, J., Swift, S. & Kuliopulos, A. Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc. Natl. Acad. Sci. USA. 99, 643–648 (2002).

    Article  CAS  Google Scholar 

  18. Brown, S.B. & Golstein, J.L. A proteolytic pathway that controls the cholesterol content of membranes, cells and blood. Proc. Natl. Acad. Sci. USA 96, 11041–11048 (1999).

    Article  CAS  Google Scholar 

  19. Grand-Perret, T. et al. SCAP ligands are potent new lipid-lowering drugs. Nat. Med. 7, 1332–1338. (2001).

    Article  CAS  Google Scholar 

  20. Hartmann, T. Cholesterol, Ab and Alzheimer's disease. Trends Neurosci. (Suppl.) 11 S45–S48 (2001).

    Article  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Peter Erhardt of the Boston Biomedical Research Institute for use of the confocal microscopy facility.

Author information

Authors and Affiliations

  1. GenPath Pharmaceuticals, 300 Technology Square, Cambridge, 02139, Massachusetts, USA

    Sylvie Vincent

  2. SelectX Pharmaceuticals, 44 Hartwell Avenue, Lexington, 02421, Massachusetts, USA

    Abraham Thomas & Bradley Brasher

  3. Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, 02139, Massachusetts, USA

    John D Benson

Authors
  1. Sylvie Vincent
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abraham Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bradley Brasher
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John D Benson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John D Benson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vincent, S., Thomas, A., Brasher, B. et al. Targeting of proteins to membranes through hedgehog auto-processing. Nat Biotechnol 21, 936–940 (2003). https://doi.org/10.1038/nbt844

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt844

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