Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Rathore, Shailendra S.; Liu, Yinghui; Yu, Haijia; Wan, Chun; Lee, MyeongSeon; Yin, Qian; Stowell, Michael H.B.; Shen, Jingshi

Liposome fusion assays-Liposome fusion and data analysis were performed as previously described Yu et al., 2019) . A standard liposome fusion reaction contained 5 mM t-SNAREs and 1.5 mM v-SNARE. In lipid-mixing assays, v-SNARE liposomes were labeled with NBD and rhodamine, and were directed to fuse with unlabeled t-SNARE liposomes with or without 5 mM Munc18-1 (Yu et al., 2019) . The samples were incubated on ice for one hour before the temperature was elevated to 37°C to initiate fusion. NBD fluorescence (excitation: 460 nm; emission: 538 nm) was measured every two minutes in a BioTek Synergy HT microplate reader. In content-mixing assays, unlabeled t-SNARE liposomes were directed to fuse with sulforhodamine B-loaded v-SNARE liposomes. Sulforhodamine B fluorescence (excitation: 565; emission: 585 nm) was measured every two minutes. At the end of the reactions, 10 μL of 10% CHAPSO was added to each sample to lyse the liposomes to obtain the maximum fluorescence. Liposome fusion data were presented as the percentage of maximum fluorescence change. The maximum fusion rate within the first 10 minutes of a liposome fusion reaction was used to represent the initial rate. Statistical significance was calculated for each figure based on at least three experiments.