Figure 4: Ubiquitin-independent, ESCRT-dependent sorting of stress-internalized EGFR onto ILVs of MVBs.

(a) Immunoprecipitation (IP) of EGFR from HeLa cell lysates and immunoblotting for ubiquitin and EGFR showed robust EGFR ubiquitination after EGF stimulation but not after exposure to UVC or cisplatin. (b) Treatment of HeLa cells with EGF 1 h post-UVC exposure induced strong EGFR ubiquitination, measured as in a, that was reduced compared with EGF alone, most likely because only 50% of EGFR was available for EGF stimulation after UVC exposure. (c) Stable PAE cell sublines expressing EGFR-wt or a ubiquitination-defective EGFR (EGFR-15KR) were exposed to UVC and incubated for 1 h with anti-EGFR-gold. Cells were fixed and processed for EM. Representative images of ultrathin sections with gold in ILVs (black arrows) and on the limiting membrane of MVBs (white arrows) from both sublines are shown. (d) Immunofluorescence shows perinuclear EGFR (green) accumulation 1 h after UVC exposure followed by recycling to the plasma membrane on subsequent p38 inhibition in both PAE EGFR-wt and -15KR cells, consistent with no role for ubiquitination in stress-induced EGFR traffic. (e) PAE EGFR-wt and -15KR cells were transfected with OA1-myc and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Ubiquitination-deficient EGFR-15KR (green) showed increased co-staining with OA1-myc (red) following EGF stimulation compared with EGFR-wt, to a similar level to that shown by UVC-internalized EGFR (-wt or -15KR). Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student’s t-test). (f) Lysates from HeLa cells transfected with Hrs or Tsg101 siRNA were immunoblotted after 72 h for Hrs, Tsg101 and tubulin to assess knockdown efficiency (left). RNAi-treated cells were exposed to UVC, incubated for 1 h with anti-EGFR-gold (arrows) and processed for EM. Ultrathin sections (right) show enlarged MVBs containing reduced numbers of EGFR-positive ILVs in Hrs and Tsg101 siRNA-treated compared with control RNAi cells. Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.