Figure 4.

GTPγS blocks the formation of COPI vesicles. (A) GTPγS inhibits the release of COPI from Golgi membranes induced by GAP. The two-stage incubation was performed with the first stage performed in the presence of GTPγS. The second-stage incubation was performed as indicated, followed by centrifugation to derive Golgi membrane (P) and supernatant (S). The distribution of COPI was assessed by immunoblotting for β-COP. (B) GTPγS inhibits the formation of COPI vesicles. A fraction (10% by volume) of the supernatant derived from the different conditions of the second-stage incubation was applied onto EM grids for immunogold labeling using antibodies direct against ɛ- or ζ-COP, followed by quantitation for vesicles with gold particles.