Presence of non–clathrin-associated AP-3 on tubulovesicular structures near, but not in, the TGN in WT melanocytic cells and of STX13-labeled tubules in Ap3d1^−/− cells. (A–D) Ultrathin cryosections of the human melanoma cell line, MNT-1, were fixed and immunogold labeled with the anti–AP-3δ antibody SA4 followed by protein A conjugated to 15-nm gold particles (AP3 15) and then antibodies to either the clathrin heavy chain (Clat.; A and B) or the TGN marker TGN46 (C and D) followed by protein A conjugated to 10-nm gold particles (10). Sections were then processed and analyzed by electron microscopy. (A) A section near a vacuolar endosome (star) emphasizing clathrin-coated vesicles, some of which also label for AP-3 (arrow). (B–D) Sections near the Golgi apparatus (GA). (B) Separate tubulovesicular structures near the Golgi are labeled for either clathrin (arrowheads) or AP-3 (arrows), but not both. (C and D) Tubulovesicular structures labeled for AP-3 (arrows) are distinct from TGN structures labeled for TGN46 (arrowheads). (E and F) Ap3d1^−/− melan-mh cells transiently transfected to express mCherry-labeled STX13 (mCh-STX13) were analyzed 48 h after transfection by live-cell spinning disk microscopy. Shown are individual frames from two representative cells documenting the presence of long STX13-containing tubules (arrowheads), similar to those observed in WT cells. Boxed regions are magnified fourfold in the insets. Scale bar, 10 µm; inset scale bar, 2 µm.