Figure 1.
HOPS stimulates trans-SNARE complex assembly between proteoliposomes but does not affect the assembly of SNAREs in detergent. (A) Schematic representation of the SNAREs used in this study. (B) Schematic representation for the topology of the SNAREs used to drive trans-SNARE complex formation in the absence of cis-SNARE complex disassembly. (C) Proteoliposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor) were incubated with HOPS (90 nM) or HOPS buffer but without Vam7p for 10 min at 27°C. All reactions contained 0.5 mM MgCl2. Triton X-100 (in RB150) or RB150 (no Triton X-100) was added where indicated, immediately followed by Vam7-3Δp (200 nM) addition. After 15 min, RIPA buffer was added, Vam3p was immunoprecipitated, and Vam3p and Nyv1p were detected by immunoblot (see Materials and Methods). % total refers to the indicated percentage of the starting proteoliposome mixture, to allow evaluation of the efficiency of SNARE coisolation. Data shown are representative of three independent experiments. See Supplemental Figure S2 for mean ± SD of three experiments.