Mechanism and function of synaptotagmin-mediated membrane apposition

Synaptotagmin-1 aggregates membranes in response to Ca 2+ . Using a variety of approaches, it was discovered that synaptotagmin-mediated membrane aggregation occurs through trans interactions between synaptotagmin molecules bound to different membranes. Synaptotagmin-regulated, SNARE-catalyzed fusio...

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Published in:	Nature structural & molecular biology Vol. 18; no. 7; pp. 813 - 821
Main Authors:	Chapman, Edwin R, Hui, Enfu, Gaffaney, Jon D, Wang, Zhao, Johnson, Colin P, Evans, Chantell S
Format:	Journal Article
Language:	English
Published:	New York Nature Publishing Group US 01-07-2011 Nature Publishing Group
Subjects:	631/45/535 631/80/313/2378 Animals Binding Sites Biochemistry Biological Microscopy Biomedical and Life Sciences Calcium - chemistry Calcium - metabolism Cell membranes Dimerization Fluorescence Resonance Energy Transfer Life Sciences Liposomes - chemistry Liposomes - metabolism Membrane Biology Membrane Fusion - physiology Membrane proteins Membranes Mice Models, Biological Molecular biology Neurotransmitters Physiological aspects Protein Interaction Mapping Protein Structure Protein Structure, Tertiary Proteins Qa-SNARE Proteins - chemistry Qa-SNARE Proteins - metabolism Rats Sensors SNARE Proteins - metabolism SNARE Proteins - physiology Synaptosomal-Associated Protein 25 - chemistry Synaptosomal-Associated Protein 25 - metabolism Synaptotagmin I - chemistry Synaptotagmin I - metabolism Synaptotagmin I - physiology United States
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Summary:	Synaptotagmin-1 aggregates membranes in response to Ca 2+ . Using a variety of approaches, it was discovered that synaptotagmin-mediated membrane aggregation occurs through trans interactions between synaptotagmin molecules bound to different membranes. Synaptotagmin-regulated, SNARE-catalyzed fusion further requires synaptotagmin to drive the assembly of nonfusogenic syntaxin and SNAP25 into fusion-competent t-SNARE heterodimers. Synaptotagmin-1 is a Ca 2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca 2+ . However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1–mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca 2+ -bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes—using non-synaptotagmin proteins—also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca 2+ -bound synaptotagmin-1 and catalyzed by SNAREs.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS E.R.C conceived of and supervised the project. E.H., J.D.G and E.R.C. designed the experiments and wrote the manuscript; E.H. conducted all the aggregation assays on syt proteins and cPLA2-C2, membrane penetration assays, and FRET-based membrane binding assays; J.D.G. conducted all the fusion assays on syt proteins and cPLA2-C2, and performed experiments for Fig. 6 with Z.W.; Z.W carried out experiments for Fig. 9; C.P.J conducted avdin-biotin mediated aggregation and fusion assays in Fig. 2d, i and Fig. 8e–h; C.S.E carried out experiments in Supplementary Fig. 8. Present address: Department of Molecular and Cellular Pharmacology, University of California, San Francisco, 600-16th street, San Francisco, CA, 94158
ISSN:	1545-9993 1545-9985
DOI:	10.1038/nsmb.2075