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Membrane protein sequestering by ionic protein–lipid interactions

Nature volume 479, pages 552–555 (2011)Cite this article

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Abstract

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.

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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.

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Acknowledgements

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.).

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Authors and Affiliations

  1. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany

    Geert van den Bogaart, Hayder Amin, Markus Dier & Reinhard Jahn

  2. Institute for Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Tammannstraße 2, 37077 Göttingen, Germany

    Karsten Meyenberg, Barbara E. Hubrich & Ulf Diederichsen

  3. Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany

    H. Jelger Risselada & Helmut Grubmüller

  4. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany

    Katrin I. Willig & Stefan W. Hell

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  1. Geert van den Bogaart
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Contributions

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.

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Correspondence to Reinhard Jahn.

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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)

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van den Bogaart, G., Meyenberg, K., Risselada, H. et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature 479, 552–555 (2011). https://doi.org/10.1038/nature10545

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