Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: The role of N-linked glycans and the unfolded protein response

We are studying endoplasmic reticulum-associated degradation (ERAD) with th e use of a truncated variant of the type I ER transmembrane glycoprotein ri bophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino-acids of the luminal domain of RI332 has been shown to interact w ith calnexin and to be a substrate for Me ubiquitin-proteasome pathway. Whe n RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics ; an initial, slow :phase of similar to 45 min was followed by a second pha se of,threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation co nsensus site had been removed (RI332-Thr) was monophasic and rapid, implyin g a role of the N-linked glycan in Me first proteolytic phase. RI332:degrad ation was enhanced when the binding of glycoproteins to calnexin was preven ted. Moreover, the truncated glycoprotein interacted with calnexin preferen tially during the first proteolytic phase, which strongly suggests that bin ding of RI332 to the lectin-like protein may result in the slow, initial ph ase of degradation. Additionally, mannose trimming appears to be required f or efficient proteolysis of RI332. After treatment of cells with the inhibi tor of N-glycosylation, tunicamycin, destruction of the truncated RI varian ts was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the pr esence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the ind uction of BiP and other ER-resident proteins. Indeed,only in drug-treated c ells could an interaction between BiP and RI332 and RI332-Thr be-detected. Induction of BiP was also evident after overexpression of murine Ire1, an E R transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results Suggest that binding of the substrate proteins to UPR-induced chaperones a ffects their half lives.