Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyrogl obulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhib itors of ER mannosidase I (but not other glycosidases) acutely suppressed C og Tg degradation and also perturbed the ERAD process for Tg reduced with d ithiothreitol as well as for gamma -carboxylation-deficient protein C expre ssed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaper ones calnexin and calreticulin to monoglucosylated oligosaccharides. Notabl y, the undegraded protein fraction remained completely microsome-associated . In pulse-chase studies, kifunensine-sensitive degradation was still inhib itable even 1 h after Tg synthesis. Intriguingly, chronic treatment with ki funensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary c ells and did not lead to significant induction of the ER unfolded protein r esponse. We hypothesize that, in a manner not requiring lectin-like activit y of calnexin/ calreticulin, the recognition or processing of a specific br anched N-linked mannose structure enhances the efficiency of glycoprotein r etrotranslocation from the ER lumen.