Figure 1.

ERdj3 is a soluble, resident ER glycoprotein. (A) Microsomes purified from Ag8.653 cells were incubated with the indicated detergents. Samples were centrifuged, and both pellets (P) and supernatants (S) were analyzed by Western blotting. (B) ERdj3 was in vitro translated in the presence of rat liver microsomes and then incubated with (+) or without (-) proteinase K or NP-40 lysing buffer as indicated. The precursor proteins that were not transported into the ER are indicated with solid arrows, whereas proteins that were translocated and protected are indicated with empty arrows. The two species of translocated ERdj3 represent signal-peptide-free ERdj3 with (a) or without (b) glycosylation. Yeast prepro α-factor was used as a positive control for translocation and glycosylation. (C) ERdj3 was in vitro translated in the absence of microsomes or immunoprecipitated from Ramos cells metabolically labeled in the presence or absence of tunicamycin (Tm). Samples in lanes 2 and 4 were further treated with Endo H before analysis by SDS-PAGE. The sample in lane 2 is overloaded.