Extended Data Fig. 9: Bulk fusion assay for the VAMP2.
a, % Fusion vs the time course data for the VAPM2 in SV like and EPC-SV vesicles. In both the cases, CDV (cytosolic domain of VAMP2) was used a negative control. In presence of excess CDV, CDV binds and occupies the VAMP2 binding domains of the t-SNAREs. Consequently, upon addition of VAMP2 reconstituted vesicles, these vesicular VAMP2s cannot bind to t-SNARE anymore and no fusion is observed. As seen, the EPC-SV vesicles, devoid of PC, fuses with the PM at a significantly lower rate than the PC containing native-like SV liposomes. b, SDS-Gel picture showing similar band intensity of VAMP2 in SV like and EPC-SV vesicles. c, Vesicle fusion assay performed between t-SNARE in PM like lipid composition vesicles (tSNARE-PM) and VAMP2 in SV like vesicles (VAMP2-PC). Subsequently, to assess the effect of EPC on fusion rate, PC was replaced with EPC in PM like vesicles containing t-SNARE. Similar rate of fusion was observed as observed in t-SNARE in PM like vesicles with PC. This confirms that the reduced fusion rate observed for SV-EPC vesicles (Fig. 2), is not because of replacing PC with EPC in the bilayer, but due to abrogation of VAMP2-PC binding. The data presented as mean ± s.e (N = 5).
