Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

Abstract

We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI 332 , containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI 332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ϳ45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI 332 in which the single used N-glycosylation consensus site had been removed (RI 332 -Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI 332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI 332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI 332 . After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI 332 and RI 332 -Thr were stabilized, despite the presence 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 induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI 332 and RI 332 -Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI 332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives. INTRODUCTIONNewly synthesized proteins of the endomembrane system and most secreted proteins of eukaryotic cells enter the secretory pathway through the translocation channel at the membrane of the endoplasmic reticulum (ER). The lumen of the ER is the site where translocated proteins assume their secondary structure and where assembly of oligomeric complexes occurs. Additionally, newly synthesized proteins undergo cotranslational and posttranslational modifications in the lumen of the ER, some of which allow transient interactions of the folding polypeptide chains with a set of ER-resident proteins (Hammond and Helenius, 1995;Leitzgen and Haas, 1998). Only after acquiring a fully folded, native conformation can proteins complete their journey through the secretory pathPresent addresses:† Dipartimento Medicina Sperimentale e Diagnostica, Sezione di Patologia Generale, Universitá di Ferrara, Ferrara, Italy; ‡ Spiegelmayr K...

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