(A) SNARE complex (500 nM) was assembled on Vam3-GST. Sec17 (20 μM), Vps33 (2.5 μM), or both were incubated with the SNARE complex for 1 hr at 30°C in SM Assay Buffer. Unbound material was washed out, then bound material was separated by SDS-PAGE and visualized with Coomassie blue. (B) The dose–response for Sec17 stimulation of Vps33 binding to SNARE complexes was assayed as in A, but Sec17 concentration was varied (0.5–20 μM) while Vps33 was held constant (2.5 μM). (C) Vps33 binding to SNARE complex with or without Sec17 was assayed as in A and B, except that Vps33 concentration was varied and protein bands were stained and quantified using SYPRO Ruby. The fractional saturation of total Vps33–SNARE complex binding was plotted vs free (total minus bound) Vps33. Fits of a one-site binding model yielded Kdobs = 300 ± 10 nM for Vps33 binding to the SNARE complex in the presence of Sec17, and Kdobs = 1.6 ± 0.10 µM without Sec17. Two-site or cooperative binding models did not substantially improve the fits. (D) To estimate the stoichiometry of SNARE–Sec17–Vps33 binding, complexes were assembled under saturation binding conditions, separated by SDS-PAGE, and analyzed using SYPRO Ruby stain. The band intensities were quantified using standard curves generated with individual purified proteins. (E) Vps33 binding is stimulated by SNARE-associated Sec17. Complexes were assayed in lanes 1 and 2 as in A. In lane 3 (Sec17 pre-bound), Sec17 was bound to SNARE complexes for 60 min at 30°C. Unbound Sec17 was washed out and 2.5 μM Vps33 was then added for an additional 60 min at 30°C. In lane 4, 20 µM BSA was substituted for Sec17. (F) Cooperativity of assembly. GST-Vps33 (500 nM) was immobilized and incubated with 20 μM Sec17, soluble SNARE complex, or both. Bound material was separated by SDS-PAGE and stained with Coomassie blue, or analyzed by immunoblot.