Setup and characterization of the in vitro fusion system. (A) Reporter systems for lipid and content mixing. RPLs (reconstituted proteoliposomes) are prepared with encapsulated content mixing FRET pair, and with the membranes doped with an orthogonal FRET pair. (B) SNARE topology of the RPLs used in this study, and soluble components added to stimulate fusion. (C–E) Characterization of the system using content mixing readout. (C) Requirement for Sly1. Reactions were set up with Q- and R-SNARE RPLs, and 3% PEG. Fusion activity was monitored for 5 min, and then Sly1 was added at time = 0 (arrows) to the indicated final concentrations. Note that fusion activity is saturated at 100 nM (Sly1). (D) Requirement for tethering. Reactions were set up with Q- and R-SNARE RPLs, with the indicated final concentrations of PEG. Fusion activity was monitored for 5 min, and then Sly1 was added to a final concentration of 250 nM. Note that at 6% and 7% PEG, some Sly1-independent fusion occurs prior to Sly1 addition. (E) Effects of the SNARE disassembly machinery. Reactions were set up with Q- and R-SNARE RPLs, and with or without PEG, Sec17, Sec18, ATP, and Sly1, as indicated. Fusion was initiated by adding Sly1. For C–E, points show mean ± SEM of three independent experiments; in many cases the error bars are smaller than the symbols. Gray lines show least-squares nonlinear fits of a second-order kinetic model.