Figure 4.

Retrotranslocation of EGFR from the ER to cytosol. (A) Cells were incubated with EGF for 3 h and the ER was OptiPrep was purified. Cytosol prepared from control cells was added as indicated to equal aliquots of ER. After a 60-min incubation at 37°3 C, the reaction mixture was centrifuged to yield pellet (P) and soluble (S) fractions. These fractions were then blotted for EGFR or Sec61β as indicated. (B) ER was incubated with control or HSP70 immunodepleted cytosol and then blotted with antibodies as indicated. (C and D) ER was preincubated with anti-Sec61β (C) or exotoxin A (D) for 30 min at room temperature before addition of cytosol. (E) Cells were subjected to cell surface biotinylation and incubated with EGF for 3 h. The ER fraction was isolated and incubated for 60 min at 37°C without or with cytosol prepared from untreated cells. The reaction mixture was centrifuged to yield pellet (P) and soluble (S) fractions. Detergent was then added to solubilize the ER fraction, and anti-EGFR was added to each fraction and the precipitates were blotted with HRP-streptavidin. The film was exposed for two different times to more clear show receptor bands in the P and S fractions. Equal aliquots of P and S fractions were also blotted for Sec61β. (F) ER fraction was incubated for 60 min at 37°C with cytosol. The soluble fraction was precipitated with anti-EGFR, and the precipitate was incubated with Endo H. The mixtures were then subjected to blotting for EGFR (lanes 1 and 2). EGFR immunoprecipitates from cell lysates from control (lanes 3 and 4) or swainsonine-treated (SW) cells (lanes 5 and 6) were digested by Endo H and blotted for EGFR. Arrows mark the 170-kDa mature receptor and a 150-kDa receptor fragment.