Fig. 4.

Phosphorylated HOPS discriminates between GTP- and GDP-charged Ypt7-tm for proteoliposome fusion. HOPS and phospho-HOPS were purified as described (12). Fusion reactions contained proteoliposomes bearing R- or QaQb-SNARE at 1:16,000 SNARE to lipid molar ratio and either Ypt7-pr (A) or Ypt7-tm (B) at a 1:8,000 protein to lipid molar ratio. R- and QaQb-proteoliposomes were separately charged with GDP or GTP and preincubated for 10 min at 27 °C. R- and QaQb-proteoliposomes were then moved to same well, mixed with 100 nM Qc, and then mixed with either 50 nM HOPS (black and green lines) or 50 nM phospho-HOPS (P-HOPS; red and blue lines). The content mixing between proteoliposomes was assayed as the FRET between luminal Cy5 and phycoerythrin for 30 min at 27 °C. Kinetics in this figure are representative of n ≥ 3 experiments; see SI Appendix, Fig. S4.