Figure 4

Comparison of the intracellular trafficking pathways of LRP10^wtand LRP10^2DXXAA. The internalization of LRP10 was evaluated in HeLa cells transfected with LRP10^wt or LRP10^2DXXAA tagged with an extracellular FLAG epitope. The cells were pre-incubated on ice to arrest endocytosis, and LRP10 molecules exposed on the cell surface were immunolabeled with antiserum directed against FLAG at 4°C. The cells were then washed and incubated at 37°C to allow internalization. Endocytosis and TGN targeting of cell surface-labeled LRP10 were evaluated after 0, 5, 10, 60, and 120-min chases at 37°C. The cells were then fixed, permeabilized, and immunostained with anti-TGN46 (green) or anti-EEA1 (blue) antibodies. The labeled cells were examined by confocal fluorescence microscopy. In cells expressing FLAG-LRP10^wt, low level of FLAG-LRP10^wt was observed at the cell surface at time 0 (A, inset, arrows). Internalized LRP10^wt (int-LRP10^wt) was observed in vesicles near the plasma membrane following a 5-min (C) and a 10-min (E) chase where it colocalized with EEA1 (C, E, insets, arrowheads). After a 60-min chase, int-LRP10^wt localized in the Golgi region and partially colocalized with the TGN marker TGN46 (G, inset, arrowheads). This colocalization was higher after a 120-min chase (I, inset, arrowhead). In cells expressing FLAG-LRP10^2DXXAA, large amounts of LRP10^2DXXAA were detected on the cell surface at time 0 (B, arrows). After a 5-min chase, int-LRP10^2DXXAA was still distributed at the plasma membrane (D, arrow) or partially colocalized with EEA1 in vesicles near the PM (D, inset, arrowheads). After a 10-min chase, int-LRP10^2DXXAA was observed in EEA1-labeled vesicles (F, inset, arrowheads). However, after a 60-min chase (H), and even a 120-min chase (J), int-LRP10^2DXXAA was still mainly observed in early endosomes (H, J, insets, arrowheads). Scale bar, 10 μm.