title: Studying protein-reconstituted proteoliposome fusion with content indicators in vitro.
abstract: In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-seconds observation period, for Ca(2+) -triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA. diaojj@gmail.com
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.forename: Minglei
authors.initials: M
authors.lastname: Zhao
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Minjoung
authors.initials: M
authors.lastname: Kyoung
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D002118:Calcium / Q000378:metabolism; D020285:Cryoelectron Microscopy; D005069:Evaluation Studies as Topic; D008055:Lipids / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology*; D008958:Models, Molecular; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012984:Software
chemical_list: D008055:Lipids; D011510:Proteolipids; D050600:SNARE Proteins; C020485:proteoliposomes; D002118:Calcium