Figure 5.

PI3P signaling regulates EGFR sorting. (A) EGF was pulsed for 10 min at 37°C in cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A). Early endosome (EE), late endosome (LE), and heavy membrane (HM) fractions (Aniento et al., 1993a) were then analyzed by Western blotting using anti-GFP antibodies. (B–D) The in vitro formation of ECVs was measured (as in Fig. 4 C) using donor early endosomes from cells expressing EGFR-GFP (as in A) in the presence or absence of ATP, wortmannin (Wort), GST-2xFYVE, or C215S mutant. Fractions containing donor early endosomes (EE) and vesicles formed in vitro were analyzed by Western blotting using antibodies against the indicated antigens. In B and D, 50 and 10% of the donor membranes (EE), respectively, were analyzed. Scanning of the lanes showed that, within the 30-min incubation of the assay, ∼10% of the EGFR present in untreated donor membranes was incorporated into newly formed ECVs. This value is somewhat lower than EGF transport to late endosomes in vivo (∼60% after 45 min; Fig. 3 C), in part because one round of vesicle formation only is reconstituted in vitro (Aniento et al., 1993a).