The Sly1 regulatory loop has a positive function in vitro. (A–D) Fusion activity of Sly1∆loop versus wild-type Sly1. Master mixes were assembled as in Fig. 3 and incubated for 5 min at 30°C. Fusion was initiated by adding (arrows) Sly1 or Sly1∆loop at the concentrations indicated in the legend adjacent to panel B. Reactions were run in the absence (A and C) or presence (B and D) of 3% PEG; and in the absence (A and B) or presence (C and D) of Sec17, Sec18 (both 100 nM), and ATP. Points show mean ± SEM from three or more independent experiments; in some cases, the error bars are smaller than the symbols. Gray lines show least-squares nonlinear fits of a second-order kinetic model. (E) Purified Sly1∆loop protein is folded. Circular dichroism spectra of wild-type Sly1 and Sly1∆loop. The spectra are normalized to account for small differences in molecular mass and concentration. A comparison of lipid and content mixing signals for the experiments in panels B and D is presented in Fig. S3.