FIGURE 4:

Antibody pull down of Qa indicates that SNARE complex formation requires HOPS, all four SNAREs, and an intact membrane. Liposomes were prepared with Ypt7 and the integral R-SNARE Nyv1. After a 30-min incubation with HOPS and the soluble Q-SNAREs, membranes were dissolved with detergent, and the proteins associated with Qa were isolated by α-Qa pull down and analyzed by immunoblot, as described in Materials and Methods. Protein inputs were 5 pmol of integrally bound R, 8 pmol each of sQa, sQb, and Qc, and 0.5 pmol of HOPS. (A–D) Eluted samples were assayed by immunoblot for R, sQb, Qc, and HOPS via its Vps16 subunit, respectively. Data represent the averages and SDs of three independent experiments.