Fig. 3. Effect of SARA on Tf endocytosis. (A) GFP-SARA (green)-transfected HEK cells were loaded with Cy3-Tf (red) at 37°C for 6, 15 or 120 minutes before the cells were chilled, washed and fixed for GFP and Cy3 visualization. At 6 minutes, Cy3-Tf was readily detectable on small endosomes in the untransfected control cells, but not in the GFP-SARA transfected cells (arrows). At 15 minutes, Cy3-Tf becomes detectable in the GFP-SARA transfected cells. After the 2-hour loading, Cy3-Tf reached a steady-state occupancy and was distributed on both early and recycling endosomes in the control cells, whereas Cy3-Tf was largely concentrated in the SARA-positive compartments of the transfected cells. Bar, 10 µm. (B) HEK cells cotransfected with GFP-SARA and Rab5S34N were loaded with Cy3-Tf for 6 minutes before fixation. The level of internalized Cy3-Tf is indistinguishable between the double-transfected cells (indicated by cytosolic GFP-SARA signal) and the neighboring untransfected cells. (C) SARA stable transfectants and HEK cells grown in 6-well dishes were incubated with ¹²⁵I-Tf at 37°C for the time indicated. The amounts of total, internalized and surface-associated radioactivity were determined. Data are plotted as described in Wiley and Cunningham (Wiley and Cunningham, 1982), such that the slope is equal to the endocytic rate constant, K_i. Results shown are from a single experiment and are representative of those obtained on three separate occasions. (D) SARA-transfected cells and HEK cells grown on coverslips were loaded for 2 hours with Cy3-Tf in serum-free medium. The cells were then fixed and the surface TfR was labeled with Cy5 by indirect immunofluorescence. Quantitative data was obtained by summing the Cy3 and Cy5 fluorescence in each field (>20 cells per field), taking the ratio and averaging over multiple fields. The ratio of Cy5/Cy3 fluorescence, the index of the surface/total TfR, was obtained from the average of three independent experiments (means±s.e.m.).

Return to article