A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins
- A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins
- Jiajie Diao; Yuji Ishitsuka; 이한기; 주철민; Zengliu Su; Salman Syed; 신년균; 윤태영; 하택집
- SNARE; vesicle fusion assay; single vesicle FRET; Content-mixing assay; lipid-mixing assay
- Issue Date
- Nature protocols
- VOL 7, NO 5, 921-934
- SNARESNARESNARESNARESNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes
our fluorescence resonance energy transfer (FRETRETRET)-based single vesicle-vesicle fusion assays for SNARESNARESNARESNARESNAREs and accessory proteins. Both lipid-mixing (with FRETRETRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRETRETRET pairs present on a DNANA hairpin that becomes linear via hybridization to a complementary DNANA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARESNARESNARESNARESNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.
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