Figure 6.

BARS mediates a mechanism by which p-coA induces COPI vesicle formation. (A) The ability of p-coA to induce the release of COPI from Golgi membrane at the second-stage incubation is lost when the membrane is washed with 3 M KCl and is restored by the addition of BARS. CHO Golgi membrane washed with 3 M KCl was used for the two-stage incubation, with the second stage using components as indicated, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (B) The release of coatomer from the more stringently washed (3 M KCl) Golgi membrane using the combination of BARS and p-coA at the second-stage incubation represents mostly COPI vesicles. The supernatant as described above in (A), derived from the coincubation of p-coA and BARS, was labeled with antibodies directed to subunits of COPI as indicated followed by immunogold EM. Bar, 50 nm. (C) The relative efficiency of COPI release from Golgi membrane induced by adding p-coA and BARS versus that by adding GAP and BARS at the second-stage incubation. The two-stage incubation system was performed using Golgi membrane washed with 3 M KCl. After the second-stage incubation that used components as indicated, the level of β-COP released into the supernatant is quantified, and then expressed as a percentage of total (represented by β-COP level in both the pellet and supernatant). The mean of this value derived from three independent experiments is shown with standard error. (D) Cargo sorting is impaired using the combination of p-coA and BARS rather than the combination of GAP and BARS. Golgi membrane was prepared from a CHO stable cell line that expresses myc-tagged KDELR and then washed with 3 M KCl for the two-stage incubation system. At the second stage, different components as indicated were added for incubation, followed by examination by EM for the level of vesicles positive for the tagged KDELR. The mean derived from three independent experiments is shown with standard error.