Figure 8.

Vti1p physically interacts with Sec17p. Spheroplasts of GWY154 cells (HA-VTI1) were lysed in the presence of Triton X-100. Solubilized proteins (0.5 mg) were incubated with purified bacterially expressed GST or GST-Sec17p (2 μg each) in buffer B (20 mM HEPES-KOH, 150 mM KOAc, 0.5 mM DTT, 0.05% Tween 20) for 1 h at 4°C and centrifuged at 15,000 × g for 15 min. The supernatant was incubated for 1 h at 4°C with 40 μl of glutathione-Sepharose 4B equilibrated with buffer B. Beads were washed three times with buffer B, and bound proteins were eluted with 50 μl of 10 mM reduced glutathione in 50 mM Tris-Cl (pH 7.5), separated by SDS-PAGE (10% acrylamide) followed by immunoblotting with anti-HA, anti-Sed5p, or anti-Kar2p antibodies. Beads that had not been incubated with yeast lysate were included as a primary antibody specificity control.