Gain-of-function Sly1 mutants alleviate the tethering requirement in vitro. (A–D) Reactions were set up as in Fig. 2, with the initial mixture containing R-SNARE and Qabc-SNARE RPLs and, as indicated for each row of panels, 0 or 3% PEG, and in the absence or presence of Sec17, Sec18 (both 100 nM), and ATP (1 mM). After a 5-min incubation, wild-type Sly1 or the indicated mutants were added (arrows) at 0, 25, 100, or 400 nM to initiate fusion. Points show the mean ± SEM from three or more 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.