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 Ca2+‐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.