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:

GTPase activity of dynamin and resulting conformation change are essential for endocytosis

Abstract

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes1,2. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear3,4. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors5. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Domain structure of dynamin showing mutants of the GTPase domain.
Figure 2: Uptake of biotinylated transferrin (green staining) into COS-7 fibroblasts, transiently overexpressing dynamin or dynamin mutants (red staining).
Figure 3: Thin-section electron microscopy of T65A-transfected COS cell.
Figure 4: Conformational changes in dynamin on GTP hydrolysis.

Similar content being viewed by others

References

  1. Urrutia, R., Henley, J. R., Cook, T. & McNiven, M. A. The dynamins: redundant or distinct functions for an expanding family of related GTPases? Proc. Natl Acad. Sci. USA 94, 377–384 (1997).

    Article  ADS  CAS  Google Scholar 

  2. van der Bliek, A. M. Functional diversity in the dynamin family. Trends Cell Biol. 9, 96–102 (1999).

    Article  CAS  Google Scholar 

  3. Warnock, D. E., HInshaw, J. E. & Schmid, S. L. Dynamin self-assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310–22314 (1996).

    Article  CAS  Google Scholar 

  4. Roos, J. & Kelly, R. B. Is dynamin really a ‘pinchase’? Trends Cell Biol. 7, 257–259 (1997).

    Article  CAS  Google Scholar 

  5. Sever, S., Muhlberg, A. B. & Schmid, S. L. Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis. Nature 398, 481–486 (1999).

    Article  ADS  CAS  Google Scholar 

  6. Poodry, C. A. & Edgar, L. Reversible alterations in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire. J. Cell Biol. 81, 520–527 (1979).

    Article  CAS  Google Scholar 

  7. Kosaka, T. & Ikeda, K. Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J. Neurobiol. 14, 207–225 (1983).

    Article  CAS  Google Scholar 

  8. Koenig, J. H. & Ikeda, K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J. Neurosci. 9, 3844–3860 (1989).

    Article  CAS  Google Scholar 

  9. van der Bliek, A. M. et al. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, 553–563 (1993).

    Article  CAS  Google Scholar 

  10. Herskovits, J. S., Burgess, C. C., Obar, R. A. & Vallee, R. B. Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122, 565–578 (1993).

    Article  CAS  Google Scholar 

  11. Damke, H., Baba, T., Warnock, D. E. & Schmid, S. L. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127, 915–934 (1994).

    Article  CAS  Google Scholar 

  12. Takei, K., McPherson, P. S., Schmid, S. L. & DeCamilli, P. Tubular membrane invaginations coated by dynamin rings are induced by GTPγS in nerve terminals. Nature 374, 186–190 (1995).

    Article  ADS  CAS  Google Scholar 

  13. Sweitzer, S. M. & Hinshaw, J. E. Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93, 1021–1029 (1998).

    Article  CAS  Google Scholar 

  14. Takei, K. et al. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131–141 (1998).

    Article  CAS  Google Scholar 

  15. Stowell, M. H. B., Marks, B., Wigge, P. & McMahon, H. T. Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring. Nature Cell Biology 1, 27–32 (1999).

    Article  CAS  Google Scholar 

  16. Takei, K., Slepnev, V. I., Haucke, V. & De Camilli, P. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nature Cell Bio. 1, 33–39 (1999).

    Article  CAS  Google Scholar 

  17. Muhlberg, A. B., Warnock, D. E. & Schmid, S. L. Domain structure and intramolecular regulation of dynamin GTPase. EMBO J. 16, 6676–6683 (1998).

    Article  Google Scholar 

  18. Prakash, B., Renault, L., Praefcke, G. J., Herrmann, C. & Wittinghofer, A. Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism. EMBO J. 19, 4555–4564 (2000).

    Article  CAS  Google Scholar 

  19. Okamoto, P. M., Tripet, B., Litowski, J., Hodges, R. S. & Vallee, R. B. Multiple distinct coiled-coils are involved in dynamin self-assembly. J. Biol. Chem. 274, 10277–10286 (1999).

    Article  CAS  Google Scholar 

  20. Pai, E. F. et al. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351–2359 (1990).

    Article  CAS  Google Scholar 

  21. Otero, A. D. Transphosphorylation and G protein activation. Biochem. Pharmacol. 39, 1399–1404 (1990).

    Article  CAS  Google Scholar 

  22. Wilson-Kubalek, E. M., Brown, R. E., Celia, H. & Milligan, R. A. Lipid nanotubes as substrates for helical crystallization of macromolecules. Proc. Natl Acad. Sci. USA 95, 8040–8050 (1998).

    Article  ADS  CAS  Google Scholar 

  23. Hopkins, C. R. & Trowbridge, I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J. Cell Biol. 97, 508–521 (1983).

    Article  CAS  Google Scholar 

  24. Vallis, Y., Wigge, P., Marks, B., Evans, P. R. & McMahon, H. T. Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis. Curr. Biol. 9, 257–260 (1999).

    Article  CAS  Google Scholar 

  25. Achiriloaie, M., Barylko, B. & Albanesi, J. P. Essential role of the dynamin pleckstrin homology domain in receptor mediated endocytosis. Mol. Cell. Biol. 19, 1410–1415 (1999).

    Article  CAS  Google Scholar 

  26. Smirnova, E., Shurland, D. L., Newman-Smith, E. D., Pishvaee, B. & van der Bliek, A. M. A model for dynamin self-assembly based on binding between three different protein domains. J. Biol. Chem. 274, 14942–14947 (1999).

    Article  CAS  Google Scholar 

  27. Grabs, D. et al. The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence. J. Biol. Chem. 272, 13419–13425 (1997).

    Article  CAS  Google Scholar 

  28. Wigge, P. et al. Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis. Mol. Biol. Cell 8, 2003–2015 (1997).

    Article  CAS  Google Scholar 

  29. Owen, D. J. et al. Crystal structure of the Amphiphysin-2 SH3 domain and its role in prevention of dynamin ring formation. EMBO J. 17, 5273–5285 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Wigge for discussions, M. Ford for discussions and assistance with immunofluorescence work, and M. Higgins for help with the expression of dynamin mutants.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Harvey T. McMahon.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Marks, B., Stowell, M., Vallis, Y. et al. GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235 (2001). https://doi.org/10.1038/35065645

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35065645

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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