Fig. 7. The UIM domain of Hrs is required for efficient retention of the EGFR at the limiting membrane of the early endosome. HeLa cells were transfected with GFP-Hrs (B), GFP-Y329/334F (C) or GFP-UIM (D), or mock-transfected (A), starved 16 hours in serum-free medium and stimulated 22 hrs post-transfection for 30 minutes with EGF. Cells were processed for immunogold-labelling on cryosections as described in Materials and methods and cryosections were double-labeled for GFP (10 nm gold) and EGFR (15 nm gold). (A) In control cells, EGFR is mainly localized on internal vesicles of endosomal vacuoles. (B) In cells overexpressing GFP-Hrs, EGFR is retained in the limiting membrane of endosomal vacuoles (arrows). (C) In HeLa cells expressing GFP-Hrs Y329/334F, EGFR is retained at the limiting membrane of endosomal vacuoles (arrows). (D) By contrast, in cells transfected with GFP-Hrs-UIM, EGFR is localized at the limiting membrane (arrows) as well as on internal vesicles (arrowheads). E, endosomal vacuole; N, nucleus; PM, plasma membrane. Scale bars: 200 nm.