Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion

Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su, Cong Ma, Jose Rizo-Rey

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNAREsynaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE–Biotin) and Cy5–streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and a-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNAREcomplex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

Original languageEnglish (US)
Pages (from-to)2014-2028
Number of pages15
JournalNature Protocols
Issue number9
StatePublished - Sep 1 2017

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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