Figure 4.

Antibody to each of the class C Vps proteins inhibits homotypic vacuole fusion. Homotypic vacuole fusion was assayed as described (30) but without cytosol. Before the preparation of the fusion assay mix, each vacuole population was incubated with 0.05 mg/ml affinity-purified antibodies either to the class C Vps proteins (Vps11p, Vps16p, Vps18p, and Vps33p), other Vps proteins (Vps5p, Vps17p, and Vps45p), or hemagglutinin (HA; epitope 12CA5). A separate reaction was treated with 60 μg/ml Gdi1p as a control for complete inhibition of fusion. One unit (U) of alkaline phosphatase activity produces 1 μmol of p-nitrophenol/min per μg BJ3505 vacuolar protein.