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Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

Nature volume 409, pages 581–588 (2001)Cite this article

Abstract

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing—the subsequent core process of fusion—is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.

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Figure 1: Label transfer from iodinated calmodulin to vacuolar membrane proteins.
Figure 2: Two-dimensional separation of proteins labelled by SASD–calmodulin.
Figure 3: Calmodulin–proteolipid interaction.
Figure 4: V-ATPase pump activity is not directly required for vacuole fusion.
Figure 5: Complex formation of V0 sectors.
Figure 6: Trans-SNARE pairing and V0 trans-complexes.

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Acknowledgements

We thank D. Gallwitz, P. Kane, R. Piper and M. Harrison for plasmids and strains; C. Baradoy for assistance; and the Boehringer Ingelheim Foundation and Deutsche Forschungsgemeinschaft (SFB446) for support.

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  1. Christopher Peters, Martin J. Bayer, Jens S. Andersen and Matthias Mann: These authors contributed equally to this work

Authors and Affiliations

  1. Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, Tübingen, 72076, Germany

    Christopher Peters, Martin J. Bayer, Susanne Bühler & Andreas Mayer

  2. Department of Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, 5230, Denmark

    Jens S. Andersen & Matthias Mann

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Peters, C., Bayer, M., Bühler, S. et al. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 409, 581–588 (2001). https://doi.org/10.1038/35054500

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