Proteoliposomes with Ypt7 and R (1:8000 and 1:16,000 molar ratio to lipids, respectively) were mixed with proteoliposomes with Ypt7 and Qa and Qb SNAREs and with 50 nM HOPS and 4 μM Qc where indicated, added either at the start of incubation or after 30 min. (A) Fusion was assayed as lumenal FRET. After 30 min, Qc was added to one sample (e, red). (B) To measure complex formation, the amount of R SNARE that was immunoprecipitated from a detergent extract with anti-Qa antibody was determined. After incubation for 33 min, samples were placed on ice and mixed with five volumes of ice-chilled modified RIPA buffer [20 mM Hepes·NaOH, pH 7.4, 150 mM NaCl, 0.2% (wt/vol) BSA, 1% (vol/vol) Triton X-100,1% (wt/vol) sodium cholate, 0.1% (wt/vol) SDS] containing RIPA buffer-washed protein A magnetic beads (ThermoFisher), 5 μM GST-R and 5 μg anti-Qa antibody. After the mix was nutated at 4°C for 2 hr, beads were washed three times with 1 mL of RIPA buffer. Proteins were eluted with 100 μL of SDS sample buffer at 95°C for 5 min. Eluates were assayed by immunoblot with antibodies to R, Qa and Vps33. For sample f, the separate proteoliposomes, Qc, and HOPS were each mixed with RIPA buffer, then combined. (C) Immunoblots for the R-SNARE were scanned from five experiments, the band intensity of sample d (HOPS and Qc added at t = 0 min) was set to 100%, and the means and standard deviations are shown.