Figure 1.

Calibration of the fusion assay. (A) Schematic representation of SNARE-dependent liposome fusion reaction. Donor liposomes (v) contain an equimolar amount of NBD-PE (green beacons) and rhodamine-PE (red diamonds) and include the v-SNARE VAMP2. Each round of fusion decreases the concentration of fluorescent phospholipid in the bilayer, decreasing quenching of NBD (green beacons) and increasing the NBD fluorescence. (B) Calibration of percent of NBD fluorescence to rounds of fusion. Various lipid mixtures were prepared from the fluorescent donor lipids mix (used to prepare donor liposomes) and the nonfluorescent acceptor lipids mix in the following proportions (acceptor:donor): 0:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1, mimicking the result (as shown in A) of 0, 0.5, 1, 2, 4, and 8 rounds of fusion of donor vesicles, respectively. These lipid mixtures were used to reconstitute VAMP2 into liposomes, and the NBD fluorescence in each set of re-isolated liposomes was expressed as a percent of the maximum fluorescence. The closed and open circles represent two independent experiments. (C) Normalized NBD fluorescence versus time of incubation. Shown is a standard fusion reaction (as described in A) measuring the increase in NBD fluorescence in which liposomes were either mixed and preincubated overnight a 4°C (closed circles), thereby allowing SNARE complexes to form before fusion at 37°C, or were not preincubated (open circles). (D) The same data as in C, now expressed as rounds of fusion of donor vesicles according to the calibration curve in B. (E) Consumption of predocked vesicles. The rounds of fusion of nonpredocked vesicles were subtracted from the rounds of fusion obtained from experiments in which predocked vesicles were used, and this difference was plotted versus time. To examine whether consumption of the predocked population behaved like a first order reaction, we represented the substrate of this reaction as 1 − [rounds of fusion (for the first round)] versus time (Inset).