Abstract
The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix1,2. Viral fusion proteins consist of a membrane-disturbing ‘fusion peptide’ and a helical bundle that pin the membranes together2,3,4. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events: formation of helical bundles by SNAREs precedes hemifusion; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion: both use a docking mechanism via helical bundles5,6 and additional peptides to destabilize the membrane and efficiently induce lipid mixing7,8,9. Our results suggest that a common lipidic intermediate3 may underlie all fusion reactions of lipid bilayers.
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Acknowledgements
We thank M. Reinhardt, A. Schmidt and V. Comte for assistance, and N. Garin and M. Allegrini for help with confocal microscopy. This work was supported by grants from DFG, FNS, HFSP and Boehringer Ingelheim.
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Supplementary information
Supplementary Figure S1
Rhodamine-phosphatidylethanolamine (Rh-PE) incorporation into isolated vacuoles. (PDF 330 kb)
Supplementary Figure S2
GTPγS blocks maturation of pro-ALP that occurs by fusion. (PDF 75 kb)
Supplementary Figure S3
Confocal images of fusion of calcein-labelled vacuoles. (PDF 41 kb)
Supplementary Figure S4
Chemical structure of calcein, sulforhodamine B, and rhodamine-phosphatidylethanolamine (Rh-PE) (PDF 6 kb)
Supplementary Figure S5
Working model on the events in vacuole fusion. (PDF 7 kb)
Supplementary Figure Legends
This file contains the legends for the Supplementary Figures S1-S5. (DOC 22 kb)
Supplementary Data
Contains a detailed description and discussion of the establishment of the rhodamine-phosphatidylethanolamine based lipid-mixing assay. In a second part, control experiments concerning the inhibitory GTPγS effect on vacuolar membrane fusion are presented and discussed. Supplementary references are provided at the end of the document. (DOC 30 kb)
Supplementary Methods
Contains a description of reagents, strains used and methods not listed in the main text and Supplementary References. (DOC 32 kb)
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Reese, C., Heise, F. & Mayer, A. Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion. Nature 436, 410–414 (2005). https://doi.org/10.1038/nature03722
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DOI: https://doi.org/10.1038/nature03722
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