Figure 4.

Inhibition of p115 binding to the Golgi apparatus inhibits transport of VSV-G to the plasma membrane. NRK cells were microinjected with a mixture of plasmids encoding Δ63 GM130 or fl GM130 and VSV-G (ts-O45-G). After expression of the proteins for 6.5 h at 39.5°C, the temperature was shifted to 31°C for 60 min in the presence of cycloheximide. After fixation with paraformaldehyde, VSV-G on the cell surface was visualized with rabbit antibodies to the lumenal domain of the VSV-G protein (A, C, and E). The cells were then permeabilized and labeled with a mAb to the cytoplasmic domain (B, D, and F) followed by secondary antibodies coupled to rhodamine (anti rabbit) and FITC (anti mouse). After 60 min at the permissive temperature, surface fluorescence was lower in cells expressing Δ63 GM130 (A–D) than in cells expressing fl GM130 (E and F) at similar expression levels. Note that the surface fluorescence is greater than the total fluorescence because polyclonal antibodies were used to detect the former and mAbs were used to detect the latter. Bar, 20 μm.