Figure 5

LRP10^2DXXAAinhibits the retrograde transport of APP from the endosome to the TGN. Internalization of APP was evaluated in HeLa cells transfected with APP₆₉₅ and pcDNA3 (A, D), HA-tagged LRP10^wt(B, E), or LRP10^2DXXAA(C, F). The cells were pre-incubated on ice to arrest endocytosis, and APP molecules exposed on the cell surface were immunolabeled with antiserum directed against APP (α-22 C11) at 4°C. Endocytosis and TGN targeting of APP were evaluated after 60-min chase periods at 37°C. Cells were then fixed, permeabilized, and immunostained with anti-HA (red) and anti-TGN46 or anti-EEA1 (blue) antibodies. The labeled cells were examined by confocal fluorescence microscopy. In cells expressing pcDNA3 and LRP10^wt-HA, internalized APP (int-APP, green) was localized in the Golgi region and partially colocalized with the TGN marker TGN46 (A, B, arrowheads). However, in cells expressing LRP10^2DXXAA-HA, internalized APP (int-APP) was distributed mainly in vesicles labeled with the endosomal marker EEA1 and LRP10^2DXXAA-HA (F, arrowheads). Arrowheads indicate structures in which APP colocalized with TGN46 or EEA1 while arrows indicate structures in which APP and TGN46 or EEA1 did not colocalize. Scale bar, 10 μm.