FIGURE 7.

Vam7p does not rescue the pah1Δ fusion defect. A, fusion reactions containing WT or pah1Δ vacuoles were treated with buffer or anti-Sec17p antibody to block SNARE priming. Fusion reactions were also treated with the recombinant 400 nm GST-Vam7p to bypass the priming block. Alk. Phos., alkaline phosphatase. B, SNARE complex formation was tested using WT and pah1Δ vacuoles. Fusion reactions were treated with anti-Sec17 antibody to inhibit SNARE priming. GST-Vam7p (400 nm) was added to bypass the priming block. A subset of reactions was treated with anti-Vam3 antibody to block the Vam7p bypass. GST-Vam7p protein complexes were isolated as described under “Experimental Procedures” and analyzed by immunoblotting using antibodies against Vam7p, Vam3p, Vps11p, and Vps33p.