In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI 332 ) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded.Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI 332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI 332 . Cell fractionation experiments indicate that RI 332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitinationdependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI 332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI 332 , its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61.From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.

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...

Abstract. Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the proteintranslocating apparatus of this organelle, were expressed in permanent HeLa cell transformants . Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RI,67) and the other consisting of the luminal 332 N112-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (tv2 = 25 h), were rapidly degraded (ti/2 < 50 min) by a nonlysosomal mechanism . The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of R1467 was monophasic (t 2 = 50 min) but the rate of degradation of R1332 molecules increased about threefold -50 min after their synthesis. Seveal pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of 81332 molecules that are not degraded during the first phase to a second degradative compartment . Thus, IKE any subcellular organelle, the ER contains proteins that permanently reside within it and function in its various biosynthetic and metabolic activities. How ever, it also contains proteins with other subcellular destinations that transit through the ER after they are inserted into its membrane or translocated into its lumen during the course oftheir synthesis on membrane-bound ribosomes (for review see Sabatini and Àdesnik, 1989). Polypeptides synthesized in the ER not only undergo various co-and posttranslational modifications within the organelle, but are also folded and in some cases oligomerized to form multisubunit proteins within the ER itself (for review see Rose and Doms, 1988;Hurtley and Helenius, 1989), apparently assisted by resident proteins which prevent the egress ofincompletely assembled products (Bole et al., 1986; deSilva et al., 1990;Siegel and Walter, 1989). when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of 81332 . On the other hand, when CCCP was added after the second phase of degradation of 81332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of 81332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded . One is the ER itself and the second is a nonlysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of 81332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca 2+ concen...

Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca 2؉ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of ␤-COP from the Golgi apparatus. We conclude that the maintenance of a Ca 2؉ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFAinduced retrograde transport from the Golgi apparatus to the ER to occur.

A soluble form of ribophorin I (RI(332)) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1)Man(5)GlcNAc(2) N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI(332)-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc(3)Man(9)GlcNAc(2) N-glycan. ER-associated degradation (ERAD) of RI(332)-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI(332)-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of alpha1,2-mannosidase activity also stabilized RI(332)-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose alpha1,2-linkage that is absent from the truncated Man(5) structure. Interestingly, though the Man(5) derivative was present in large amounts in the total protein pool, the two major species linked to RI(332)-3HA shortly after synthesis consisted of Glc(1)Man(5 )and Man(4), being replaced by Man(4 )and Man(3) when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI(332)-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an alpha1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI(332-)3HA in the glycosylation-defective cell line, MadIA214.