Figure 3.

HOPS release is enhanced in vam7Δ vacuoles. BJ3505 wt or vam7Δ vacuoles (12 μg) were incubated in reaction buffer for 90 min in the absence or presence of ATP (24). Gdi1p (64 μg/ml) was added where indicated. One sample was incubated in a 20-fold diluted reaction volume. The reactions were then diluted 5-fold with 10 mM Pipes/KOH (pH 6.8) and 200 mM sorbitol, and the vacuoles were sedimented by centrifugation (14,000 × g, 5 min, 4°C). Proteins in the supernatant were precipitated as described in Fig. 1 and were analyzed by SDS/PAGE and immunoblotting with antibodies to Vam6p. Bands were quantified by laser densitometry.