FIGURE 4.

Phosphorylated-HOPS (P-HOPS) only stimulates SNARE-dependent proteoliposome fusion when proteoliposomes bear Ypt7p. A, scheme for the preparation of HOPS and P-HOPS. B, immunoblot for Vps41p of HOPS and P-HOPS preparations. C, lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). Proteoliposomes had no phosphoinositides or Ypt7p (−PIPs − Ypt7p), phosphoinositides and no Ypt7p (+ PIPs − Ypt7p), or Ypt7p (400 nm for acceptor proteoliposomes and 50 nm for donor proteoliposomes) and no phosphoinositides (− PIPs + Ypt7p), as indicated. All reactions contained 1.5 mm MgCl₂ and 1 mm ATP. HOPS and P-HOPS were added at the indicated concentrations. Vam7p (350 nm), Sec17p (1.2 μm), and Sec18p (1.4 μm) were added to all tubes at time 0, which followed a 10-min preincubation of the plate at 27 °C. D, lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). All reactions contained 0.5 mm MgCl₂. P-HOPS was added to a mixture of donor and acceptor liposomes 10 min before HOPS, all on ice. Vam7 (300 nm) was added at time 0, which followed a 10-min preincubation of the plate at 27 °C. Results are the mean of three independent experiments ± S.D.