Abstract
Previously we showed that fast Ca(2+)-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle-vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca(2+)-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca(2+)-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca(2+)-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca(2+)-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca(2+)-triggered fusion and inhibition of spontaneous fusion.
Keywords:
SNARE; complexin; membrane fusion; neurotransmitter release; synaptic vesicle fusion; synaptotagmin.
Copyright © 2014, Lai et al.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adaptor Proteins, Vesicular Transport / chemistry
-
Adaptor Proteins, Vesicular Transport / genetics
-
Adaptor Proteins, Vesicular Transport / metabolism
-
Animals
-
Biological Assay
-
Biological Transport
-
Calcium / chemistry*
-
Calcium / metabolism
-
Escherichia coli / genetics
-
Escherichia coli / metabolism
-
Exocytosis
-
Gene Expression
-
Membrane Fusion*
-
Models, Biological
-
Mutation
-
Nerve Tissue Proteins / chemistry
-
Nerve Tissue Proteins / genetics
-
Nerve Tissue Proteins / metabolism
-
Neurons / chemistry
-
Neurons / cytology
-
Neurons / metabolism
-
Protein Binding
-
Protein Refolding
-
Protein Structure, Tertiary
-
Rats
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / genetics
-
Recombinant Proteins / metabolism
-
Synaptic Vesicles / chemistry*
-
Synaptic Vesicles / metabolism
-
Synaptosomal-Associated Protein 25 / chemistry
-
Synaptosomal-Associated Protein 25 / genetics
-
Synaptosomal-Associated Protein 25 / metabolism
-
Synaptotagmin I / chemistry
-
Synaptotagmin I / genetics
-
Synaptotagmin I / metabolism
-
Syntaxin 1 / chemistry*
-
Syntaxin 1 / genetics
-
Syntaxin 1 / metabolism
-
Vesicle-Associated Membrane Protein 2 / chemistry*
-
Vesicle-Associated Membrane Protein 2 / genetics
-
Vesicle-Associated Membrane Protein 2 / metabolism
Substances
-
Adaptor Proteins, Vesicular Transport
-
Nerve Tissue Proteins
-
Recombinant Proteins
-
Snap25 protein, rat
-
Stx1a protein, rat
-
Synaptosomal-Associated Protein 25
-
Synaptotagmin I
-
Syntaxin 1
-
Syt1 protein, rat
-
Vamp2 protein, rat
-
Vesicle-Associated Membrane Protein 2
-
complexin I
-
Calcium