title: A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.
abstract: SNARE (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 (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) 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 SNARE 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.
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Illinois, USA.
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishitsuka
authors.forename: Hanki
authors.initials: H
authors.lastname: Lee
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
authors.forename: Zengliu
authors.initials: Z
authors.lastname: Su
authors.forename: Salman
authors.initials: S
authors.lastname: Syed
authors.forename: Yeon-Kyun
authors.initials: YK
authors.lastname: Shin
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Yoon
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008561:Membrane Fusion / Q000502:physiology*; D009474:Neurons / Q000737:chemistry; D050600:SNARE Proteins / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D008051:Lipid Bilayers; D050600:SNARE Proteins