A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

Title
A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins
Authors
Jiajie DiaoYuji Ishitsuka이한기주철민Zengliu SuSalman Syed신년균윤태영하택집
Keywords
SNARE; vesicle fusion assay; single vesicle FRET; Content-mixing assay; lipid-mixing assay
Issue Date
2012-05
Publisher
Nature protocols
Citation
VOL 7, NO 5, 921-934
Abstract
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.
URI
http://pubs.kist.re.kr/handle/201004/43785
ISSN
17542189
Appears in Collections:
KIST Publication > Article
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