Figure 8. Staging of Sec17 rescue in cell-free assays of vacuole fusion.

(A) Master vacuole fusion reactions were assembled under no-ATP ‘bypass’ conditions (Figure 3—figure supplement 1), and pre-incubated for 25 min at 27°C. Fusion was then initiated (t₀) by adding Qc-wt to 20 nM final concentration. At the indicated time points, an aliquot was withdrawn from the master reaction and added to a tube containing the indicated inhibitor (or placed on ice), and incubated for the duration of the experiment. At t = 70 min, each reaction aliquot was assayed for content mixing. (B) Fusion reactions assembled under no-ATP ‘bypass’ conditions were pre-incubated for 10 min at 27°C. At t = −15 min, 75 nM Qc-3∆ was added and the reactions were incubated for an additional 15 min. Fusion was initiated by adding Sec17 (300 nM). At the indicated time points, an aliquot was withdrawn from the master reaction and added to a tube containing the indicated inhibitor (or placed on ice), and incubated for the duration of the experiment. At t = 70 min, each reaction aliquot was assayed for content mixing. For both panels, each point indicates mean + s.e.m. for two to six independent experiments.