FIGURE 5.

Isolation of the prebudding complex. The ER-enriched membrane fractions prepared from the cells expressing 3HA-SVP26 were incubated with GST-Sar1 and the Sec23-Sec24 complex in the presence of either nonhydrolyzable GTP analog GMPPNP or GDP analog GDPβS as described (4). Membranes were solubilized with digitonin, and proteins bound to GST-Sar1 were collected on the glutathione-Sepharose beads. The input lanes contain 1.25% of the solubilized membranes, and the bound lanes contain the whole materials eluted from the glutathione beads. Sec22 and Sec61 were monitored as a positive and negative control of the experiment, respectively.