Sorting signals bound by AP-3 are crucial for the correct targeting of DG proteins in MEG-01 cells. AP-3 partially colocalizes with Rab38 in MKs and MEG-01 cells. (A-B) A single mutation in the LAMP2 cytosolic tail sorting signal (Y/A LAMP2: YEQF into AEQF) is sufficient to mistarget Y/A LAMP2-GFP to the plasma membrane in MEG-01 cells. Live cells were visualized by spinning-disk confocal fluorescence microscopy. (C-D) Similarly, mutation of the VMAT2 cytosolic tail sorting signal (IL/AA VMAT2: EEKMAIL into EEKMAAA) causes mistrafficking of the mutant protein to the plasma membrane in MEG-01 cells. Live cells were visualized by spinning-disk confocal fluorescence microscopy. (E-E′) Primary MKs were fixed and immunostained with antibodies against AP-3, Rab38, and clathrin, and imaged by spinning-disk confocal fluorescence microscopy. (E′) Close-up view of individual structures allows observation of colocalization of AP-3 and Rab38 (merge panel, MOC = 0.34 ± 0.01, n = 7 cells), whereas clathrin is also present in many of these structures (Rab38 and clathrin MOC = 0.33 ± 0.02, n = 7 cells). (F-F′) MEG-01 cells were fixed and immunostained with antibodies against AP-3, Rab38, and clathrin and imaged by spinning-disk confocal fluorescence microscopy. (F′) Similarly to the results obtained with MKs, AP-3 and Rab38 colocalize in structures that in many cases contain clathrin (Rab38 and AP-3 MOC = 0.32 ± 0.02, n = 4 cells; Rab38 and clathrin MOC = 0.33 ± 0.03, n = 4 cells). Bars represent 5 μm.