Figure 2.
HOPS complex proofreads the 0-layer in the presence of ATP. Vacuoles were assayed for fusion in reactions where the priming block by α-Sec17p was bypassed by added Vam7p (Thorngren et al., 2004). Reactions (30 μl) contained vacuoles from BJ3505 and DKY6281 (3 μg each) in 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 150 mM KCl, 6 mM MgCl2, 10 μM CoA, 1 mM ATP, 1 mg/ml creatine kinase, 29 mM creatine phosphate, 815 nM purified IB2, and 209 nM affinity-purified α-Sec17. After 5 min on ice, HOPS and either Vam7p (A) or Vam7Q283Rp (B) was added at the indicated concentrations. Fusion was measured after 90 min at 27°C. Fusion data are from three independent reactions, standardized as a percentage of the maximum fusion signal obtained with each Vam7p addition, which was 4.78 ± 0.72 U (A) and 1.38 ± 0.33 U (B), mean ± SD.