FIG. 5.

Impaired ERAD in Perk-deficient cells. A: Retrotranslocation of CTA1 subunit in PERK-suppressed cells. AD293 cells carrying either empty vector or DNPerk were incubated with cholera holotoxin for 90 min. Cells were harvested and separated into cytosolic (C) and ER (ER) fractions and then immunoblotted for calnexin (ER marker), tubulin (cytosolic marker), CTA, or CTA1. Extent of retrotranslocation was quantified by expressing cytosolic CTA1 (normalized to tubulin) as a percentage of ER-retained CTA1 (normalized to calnexin). The percent retrotranslocation in PERK-suppressed cells normalized to empty vector is indicated underneath the immunoblot panels. B: Protein extracts from AD293 cells transfected with DNPerk or empty vector were immunoblotted for ERp72. Ablation of Perk in AD293 cells resulted in a 3.3-fold increase in ERp72. C, lane 1: V5-tagged Ins⁺-KDEL was expressed in AD293 cells and was immunoprecipitated and immunoblotted with anti-V5 antibody. Arrowhead indicates V5-tagged Ins⁺-KDEL. Lane 2: Untransfected control. Lanes 3 and 4: Ins⁺-KDEL construct was transfected into AD293 cells and lysates were immunoprecipitated with anti-V5 and then immunoblotted with the FK2 antibody that recognizes mono- and polyubiquitylated species but not free ubiquitin. Molecular weight markers (kd) are shown to the right of the blot. D: AD293 cells were cotransfected with V5-tagged wild-type proinsulin-KDEL (Ins⁺-KDEL) and empty vector (Vector) or DNPerk. Lysates were immunoblotted with the anti-V5 antibody. Arrowhead indicates V5-tagged proinsulin. A 3.6-fold increase in the retention of Ins⁺-KDEL in the PERK-suppressed cells compared with the empty-vector cotransfected controls was noted. The multiple higher–molecular weight bands (arrow) are ubiquitylated species as shown in C.