Figure 3.

HOPS does not promote fusion-dependent lysis with altered 0-layer SNARE complexes. Reactions (90 μl) containing 9 μg each of vacuoles from a BJ3505 derivative bearing lumenal soluble GFP (Starai et al., 2007) and from DKY6281 were incubated at 27°C under ATP-free conditions (see Materials and Methods) with the following modifications: BSA was used at 3 mg/ml, and reactions contained 0.1× protease inhibitor cocktail (50× stock: 13 μg/ml leupeptin, 25 mM 1,10-phenanthroline, 25 μg/ml pepstatin A, and 5 mM Pefabloc SC). This concentration of protease inhibitor does not inhibit the proteolytic activation of proPho8p during fusion. Where indicated, HOPS was added to 15.8 nM. After 45 min, reactions were gently mixed and placed on ice; 30 μl was withdrawn to measure fusion (black bars) via Pho8p activity, and 30 μl was assayed for vacuolar lysis (white bars) via centrifugation (5200 × g, 6 min, 4°C) and assay of GFP fluorescence in pellets and supernatants, as described previously (Starai et al., 2007).