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Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles

Nature Cell Biology volume 2, pages 42–49 (2000)Cite this article

Abstract

Here, to study lipid–protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

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Figure 1: Photoactivatable lipids: [3H]photocholesterol and [3H]photophosphatidylcholine.
Figure 2: Characterization of photocholesterol interactions with proteins and lipids.
Figure 3: Photoaffinity labelling of MDCK cells with photoactivatable lipids reveals specific binding of photocholesterol to caveolin/VIP21 in vivo .
Figure 4: Photoaffinity labelling of PC12 clone 251 and clone 27 cells.
Figure 5: Identification of photocholesterol-labelled proteins of PC12 clone 251 cells.
Figure 6: Specific cholesterol binding to synaptophysin in SLMVs/synaptic vesicles and their donor membranes.
Figure 7: The effect of cholesterol depletion and replenishment on PC12 cell SLMVs.
Figure 8: Cholesterol–synaptophysin interaction and the biogenesis of SLMVs.

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Acknowledgements

We thank A. Bosserhoff and R. Frank for mass spectrometry of V-ATPase c; K. Burger for help with photocholesterol synthesis; R. Sandhoff and F. Wieland for mass spectrometry of photocholesterol; A. Schmidt for advice on analysis of PC12 cell SLMVs; and P. Rosa and J. Meldolesi for PC12 clone 27 cells. This work was supported by grants from the DFG (SFB 317, C2 to W.B.H.; SFB 474 to F.F.), the EC (ERB-FMRX-CT96-0023 and ERBBIO4CT960058 to W.B.H.) and the FCI (to W.B.H.).

Correspondence and requests for materials should be addressed to C.T. or W.B.H.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology.

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  1. Matthew J. Hannah

    Present address: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK

Authors and Affiliations

  1. Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, Heidelberg, D-69120, Germany

    Christoph Thiele, Matthew J. Hannah & Wieland B. Huttner

  2. Max-Planck-Institute of Molecular Cell Biology and Genetics,, Pfotenhauerstrasse 110, Dresden, D-01307, Germany

    Christoph Thiele, Matthew J. Hannah & Wieland B. Huttner

  3. Department of Biochemistry, University of Mainz, Becherweg 30, Mainz, D-55099, Germany

    Falk Fahrenholz

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Thiele, C., Hannah, M., Fahrenholz, F. et al. Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat Cell Biol 2, 42–49 (2000). https://doi.org/10.1038/71366

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