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. 2009 Apr 21;284(24):16118–16125. doi: 10.1074/jbc.M109.000737

FIGURE 2.

FIGURE 2.

Recombinant Yck3p inhibits Ypt7p-independent reconstituted proteoliposome fusion and directly phosphorylates the Vps41p of pure HOPS. A, lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). All reactions contained 1 mm MgCl2 and 0.5 mm ATP. HOPS (66.3 nm) and Yck3p (0.45–7.15 μm) were added as indicated. Vam7p (300 nm) was added to all wells at time 0, which followed a 10-min preincubation of the plate at 27 °C. B, selected reactions from panel A were incubated with 0.4% SDS at 90 °C for 5 min followed by SDS-PAGE and immunoblot for Vps41p. C, lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). Reactions without ATP contained 0.5 mm MgCl2, and reactions with ATP contained 1 mm MgCl2 and 0.5 mm ATP. Yck3p (7.15 μm) and HOPS (66.3 nm) were added as indicated. Vam7p (300 nm) was added to all tubes 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.