Figure 7.

Rab25 inhibits transcytosis and apical recycling in adenovirus-transfected MDCK cells. (A) MDCK cells stably expressing the pIgR and the tetracycline-regulated transactivator were either mock-infected (triangles) or infected with recombinant adenovirus (squares and circles) encoding either wild-type Rab25 (A–C) or the Rab25T26N (D–F) for 18 h at 37°C. Expression of the recombinant proteins was confirmed by Western blot (B and E, insets). Infections were performed either in the absence of doxycycline (circles) or the presence of 20 ng/ml doxycycline (squares) to repress Rab25 synthesis. Cells were loaded with [¹²⁵I]-dimeric IgA from the basolateral medium for 10 min at 37°C. Filters were then washed, and fresh medium was added to apical and basolateral chambers and incubated for the times indicated. Apical media (containing transcytosed ligand, filled symbols) and basolateral media (recycled ligand, open symbols) were harvested at each time point, and cells were collected after the last time point. Data are the average of two determinations and are expressed as a percentage of total internalized cpm. Triangles indicate mock-infected; circles, − doxycycline; squares, + doxycycline. (B and E) Fraction of internalized IgA remaining inside the cells at the end of the experiment shown in A and D, respectively. (C and F) Apical recycling. Ligand was internalized from the apical surface for 30 min at 37°C. Cells were then cooled to 4°C and washed, and surface-bound ligand was removed by incubation with trypsin (10 μg/ml) for 1 h at 4°C. Trypsinization was stopped by washing with cold medium containing soybean trypsin inhibitor (50 μg/ml). Filters were then placed in warm medium, and ligand was recovered from the apical (recycled) or basolateral (transcytosed) medium at the times indicated. Apical recycling was assayed in both (C) Rab25-infected and (E) Rab25T26N-infected cells. Filled symbols indicate apical recycling; open symbols, apical-to-basolateral transcytosis: triangles, mock-infected; circles, − doxycycline; squares, + doxycycline. All data represent trichloroacetic acid-precipitable counts (typically 97% of internalized ligand was trichloroacetic acid-precipitable) and are representative of at least three separate experiments.