Skip to main content

Thank you for visiting 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:

Membrane protein sequestering by ionic protein–lipid interactions


Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A1, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis2,3. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein–lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.

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

Access options

Buy this article

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

Figure 1: PIP2 is the predominant inner-leaflet lipid in roughly 73-nm-sized microdomains.
Figure 2: Confocal microscopy of syntaxin-1A domains in artificial membranes.
Figure 3: Removal of PIP2 reduces syntaxin-1A clustering in PC12 cells.
Figure 4: Simulations of the dynamic and amorphous PIP2/syntaxin-1A microdomains.

Similar content being viewed by others


  1. Jahn, R. & Scheller, R. H. SNAREs – engines for membrane fusion. Nature Rev. Mol. Cell Biol. 7, 631–643 (2006)

    Article  CAS  Google Scholar 

  2. Aoyagi, K. et al. The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters. J. Biol. Chem. 280, 17346–17352 (2005)

    Article  CAS  Google Scholar 

  3. Lang, T. et al. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J. 20, 2202–2213 (2001)

    Article  CAS  Google Scholar 

  4. McLaughlin, S., Wang, J., Gambhir, A. & Murray, D. PIP(2) and proteins: interactions, organization, and information flow. Annu. Rev. Biophys. Biomol. Struct. 31, 151–175 (2002)

    Article  CAS  Google Scholar 

  5. McLaughlin, S. & Murray, D. Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605–611 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Wen, P. J., Osborne, S. L. & Meunier, F. A. Dynamic control of neuroexocytosis by phosphoinositides in health and disease. Prog. Lipid Res. 50, 52–61 (2011)

    Article  CAS  Google Scholar 

  7. Milosevic, I. et al. Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J. Neurosci. 25, 2557–2565 (2005)

    Article  CAS  Google Scholar 

  8. Hay, J. C. & Martin, T. F. Phosphatidylinositol transfer protein required for ATP-dependent priming of Ca2+-activated secretion. Nature 366, 572–575 (1993)

    Article  ADS  CAS  Google Scholar 

  9. James, D. J., Khodthong, C., Kowalchyk, J. A. & Martin, T. F. Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion. J. Cell Biol. 182, 355–366 (2008)

    Article  CAS  Google Scholar 

  10. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001)

    Article  CAS  Google Scholar 

  11. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)

    Article  ADS  CAS  Google Scholar 

  12. Sieber, J. J. et al. Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317, 1072–1076 (2007)

    Article  ADS  CAS  Google Scholar 

  13. Williams, D., Vicôgne, J., Zaitseva, I., McLaughlin, S. & Pessin, J. E. Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane. Mol. Biol. Cell 20, 4910–4919 (2009)

    Article  CAS  Google Scholar 

  14. Denisov, G., Wanaski, S., Luan, P., Glaser, M. & McLaughlin, S. Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results. Biophys. J. 74, 731–744 (1998)

    Article  ADS  CAS  Google Scholar 

  15. Kweon, D. H., Kim, C. S. & Shin, Y. K. The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study. Biochemistry 41, 9264–9268 (2002)

    Article  CAS  Google Scholar 

  16. Lam, A. D., Tryoen-Toth, P., Tsai, B., Vitale, N. & Stuenkel, E. L. SNARE-catalyzed fusion events are regulated by syntaxin1A-lipid interactions. Mol. Biol. Cell 19, 485–497 (2008)

    Article  CAS  Google Scholar 

  17. Murray, D. H. & Tamm, L. K. Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol. Biochemistry 48, 4617–4625 (2009)

    Article  CAS  Google Scholar 

  18. Bacia, K., Schuette, C. G., Kahya, N., Jahn, R. & Schwille, P. SNAREs prefer liquid-disordered over “raft” (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279, 37951–37955 (2004)

    Article  CAS  Google Scholar 

  19. Carvalho, K., Ramos, L., Roy, C. & Picart, C. Giant unilamellar vesicles containing phosphatidylinositol(4,5)bisphosphate: characterization and functionality. Biophys. J. 95, 4348–4360 (2008)

    Article  ADS  CAS  Google Scholar 

  20. Levental, I. et al. Calcium-dependent lateral organization in phosphatidylinositol 4,5-bisphosphate (PIP2)- and cholesterol-containing monolayers. Biochemistry 48, 8241–8248 (2009)

    Article  CAS  Google Scholar 

  21. Christian, D. A. et al. Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding. Nature Mater. 8, 843–849 (2009)

    Article  ADS  CAS  Google Scholar 

  22. Kaiser, H. J. et al. Order of lipid phases in model and plasma membranes. Proc. Natl Acad. Sci. USA 106, 16645–16650 (2009)

    Article  ADS  CAS  Google Scholar 

  23. Laage, R., Rohde, J., Brosig, B. & Langosch, D. A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins. J. Biol. Chem. 275, 17481–17487 (2000)

    Article  CAS  Google Scholar 

  24. Sieber, J. J., Willig, K. I., Heintzmann, R., Hell, S. W. & Lang, T. The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane. Biophys. J. 90, 2843–2851 (2006)

    Article  ADS  CAS  Google Scholar 

  25. Marrink, S. J., Risselada, H. J., Yefimov, S., Tieleman, D. P. & de Vries, A. H. The MARTINI forcefield: coarse grained model for biomolecular simulations. J. Phys. Chem. B 111, 7812–7824 (2007)

    Article  CAS  Google Scholar 

  26. Yesylevskyy, S., Schafer, L. V., Sengupta, D. & Marrink, S. J. Polarizable water model for the coarse-grained Martini force field. PLoS Comp. Biol. 6, e1000810 (2010)

    Article  ADS  Google Scholar 

  27. Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K. & Kusumi, A. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 1071–1082 (2002)

    Article  CAS  Google Scholar 

  28. van den Bogaart, G. & Jahn, R. Counting the SNAREs needed for membrane fusion. J. Mol. Cell Biol. 3, 204–205 (2011)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

Download references


We thank M. Holt, G. Bunt, F. S. Wouters and C. Eggeling for advice, and V. Haucke and S. Joo for the red-fluorescent-protein-tagged synaptojanin-1 construct. G.v.d.B. is financed by the Human Frontier Science Program. This work was supported by the US National Institutes of Health (P01 GM072694, to R.J.) and the Deutsche Forschungsgemeinschaft (SFB803, to K.M., H.J.R., U.D., H.G. and R.J.).

Author information

Authors and Affiliations



G.v.d.B. and R.J. designed the experiments and wrote the paper. K.M., B.E.H. and U.D. synthesized the peptides. H.J.R. and H.G. performed the simulations. K.I.W. and S.W.H performed the STED microscopy. H.A. and M.D. contributed to the protein purification, immunofluorescence and microscopy. G.v.d.B. performed all other experiments. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Reinhard Jahn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Methods, Supplementary Figures 1-15 with legends, legend for Supplementary Movie 1 and additional references. (PDF 1455 kb)

Supplementary Movie 1

The movie shows a simulation of the dynamic and amorphous PIP2-syntaxin-1A microdomains - see Supplementary information file for full legend. (MPG 23880 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

van den Bogaart, G., Meyenberg, K., Risselada, H. et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature 479, 552–555 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


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