Mannosidase action, independent of glucose trimming, is essential for proteasome-mediated degradation of unassembled glycosylated Ig light chains

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acc eptor sites into the variable domain of the murine Ig L chain kappa (NS1) w hich is unfolded in unassembled molecules. We investigated the fate of kapp a (NS1) glycosylated at position 70 (kappa 70) and of a double mutant (kapp a 18/70) in stably transfected HeLa cells. Degradation of both chains was i mpaired by lactacystin, a specific inhibitor of the proteasome. The mannosi dase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromyc in. In contrast, ER glucosidase inhibitors dramatically accelerated the deg radation of the chains when added either pre- or posttranslationally. The a ccelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide , too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappa (NS1) chains. We conclude that ER mannosidases and pr oteasome activities, but not glucose trimming (and therefore, most likely n ot the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glyco proteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.