Figure 5.

Syt1-RQ and Syt1-RA mutants fail to promote Ca²⁺-triggered lipid mixing in a reconstituted SUV–GUV fusion assay. t-SNARE-GUVs (14 nmol lipid, 14 pmol Syntaxin1/SNAP25) were mixed with ³H-labeled v-SNARE-SUVs (2.5 nmol lipid, 8.3 pmol Synaptobrevin2, 3.1 pmol Syt1) containing either Syt1-wt or Syt1–R398/399Q or Syt1-RA in the presence and absence of 6 μm CpxII in a final volume of 100 μl. The increase of Atto488 fluorescence was monitored. After 2 min at 37°C, Ca²⁺ was added to a final concentration of 100 μm and the measurement continued for another 2 min. Values were normalized to the maximum fluorescent signal after detergent lysis. Error bars indicate SEM. n = 3.