Figure 3. Phosphomimetic VAMP8 reduces in vitro ensemble vesicle fusion.

1. Schematic of the in vitro ensemble content‐mixing assay. v‐SNARE vesicles (VAMP8) loaded with content dye and unlabeled t‐SNARE vesicles (SNAP23 and STX3).
2. Reduced kinetics of content‐mixing was observed with VAMP8Glu (T47E, T53E, S54E) compared to wild‐type VAMP8 (n = 3). As a control (black line), the content‐mixing assay was performed by combining v‐SNARE vesicles (VAMP8) loaded with dye with unlabeled t‐SNARE vesicles prepared without SNAP23 and STX3. The slight steady increase of the control is due to a small amount of content dye leakage and consequent dequenching of the dyes.