Figure 7.

Sec17p release depends on PI(4,5)P₂. Vacuoles equivalent to four fusion reactions were preincubated with the indicated amounts of (A) neomycin or (B) antibodies to PI(4,5)P₂ as in Figure 1 (without cytosol). A control sample received no phosphoinositide ligands, but EDTA was added to prevent ATP hydrolysis and thereby block Sec17p release. The samples were supplemented to yield standard fusion conditions (minus cytosol) and incubated at 27°C for 15 min. Aliquots equivalent to three standard reactions (18 μg of vacuoles) were withdrawn, and the vacuoles were reisolated by centrifugation and assayed for bound Sec17p by SDS-PAGE and Western blotting. The remaining aliquots were incubated for further 55 min and used to assay fusion. Complete inhibition of fusion occurred at the same concentrations as in Figure 1 ( i.e., with 60 μM antibody and 300 μM neomycin).