Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machineryR. Venkat Kalyana Sundaram, Atrouli Chatterjee, Manindra Bera, Kirill Grushin, Aniruddha Panda, Feng Li, Jeff Coleman, Seong Lee, Sathish Ramakrishnan, Andreas M. Ernst, Kallol Gupta, James E. Rothman, Shyam S. Krishnakumar
Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca 2+ . Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+ -triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18–Syntaxin–VAMP2 “template” complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.