Figure 2.

Reconstitution of GEF-driven Ypt7–REP activation and membrane fusion. A, GEF-mediated activation of prenylated Ypt7 in fusion. Top panels, 100 nm Mon1–Ccz1 complex was added to proteoliposomes and increasing amounts of prenylated Ypt7–REP and preincubated for 15 min at 27 °C before addition of the fusion mix. Bottom panels, 500 nm GDI was added 10 min after starting the nucleotide exchange. B, EDTA-driven activation of prenylated Ypt7 in fusion. 2 mm EDTA and, after 10 min incubation, 3 mm MgCl₂ were used to drive nucleotide exchange. Fusion was measured in the absence (top panels) or presence (bottom panels) of 500 nm GDI as in A. Curves shown for content mixing assays are representatives of at least three independent experiments, using differing amounts of Ypt7, which led to the same overall result.