Figure 2. Stimulation of fusion by Sec18 does not require ATP hydrolysis, while Sec18-mediated disassembly of cis-SNARE complexes requires ATP hydrolysis.
(A) The fusion of Ypt7(GTP):R-SNARE proteoliposomes with Ypt7(GTP):QaQb-SNARE proteoliposomes with added Qc-SNARE and HOPS is stimulated by Sec17, Sec18, and ATP or ATPγS. (B) ATP hydrolysis is required for 4-SNARE proteoliposome fusion. Proteoliposomes bearing Ypt7:GTP, each of the four vacuolar SNAREs at a 1:32,000 molar ratio to lipids, and either Cy5-streptavidin or biotinylated-phycoerythrin were prepared, mixed, and assayed for fusion in the presence of HOPS, Sec17, Sec18 and ATP (filled circles), ATPγS (open squares), or without addition of adenine nucleotide (open triangles), as described in Materials and Methods. The data shown in this and the following figures were typical of 3 repeat experiments; means and standard deviations for each experiment are presented in the corresponding Supplementary Figures.
DOI: http://dx.doi.org/10.7554/eLife.26646.003
Figure 2—figure supplement 1. Average and standard deviations of fusion after 30 min for triplicate assays as in Figure 2, relative to the maximal fusion condition.
