The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human ␣ 1 -antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded ␣ 1 -antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded ␣ 1 -antitrypsin for degradation by the proteasome. The endoplasmic reticulum (ER)1 functions as the intracellular site where nascent polypeptides enter the central vacuolar system (1) and fold into their correct functional conformation (2), which is dictated by the primary amino acid sequence (3). Quality control machinery resident to that compartment facilitates the selective elimination of incompletely folded proteins to ensure that only functional molecules are deployed to distal sites (4, 5). As such, the role of conformation-based quality control is fundamental to normal cell physiology.Although initially unexpected, it is now recognized that the 26 S proteasome, a constituent of the cytosol (6), is responsible for the degradation of many ER-situated proteins (7-10). Indeed, the cytoplasmic delivery of proteasomal substrates has been reported (11)(12)(13)(14). As yet, however, the molecular basis by which proteins of the ER are selected for proteasomal degradation remains unclear, although the molecular chaperone calnexin, which binds monoglucosylated oligosaccharides (15), has been implicated as a possible participant in the sorting process (16,17).The molecular pathogenesis of several human diseases, including cystic fibrosis, familial hypercholesterolemia, and a heritable form of pulmonary emphysema, are caused, in part, by the participation of conformation-based quality control factors (for reviews, see Refs. 18 and 19). The latter disorder is caused by ...

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In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded ␣ 1 -antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.In the endoplasmic reticulum (ER), 1 an assortment of molecular chaperones and folding enzymes facilitate the conformational maturation of newly synthesized polypeptides destined for deployment to the cell surface as biologically active proteins (1). In recent years, a picture has emerged that describes how asparagine-linked glycosylation, in combination with several independently acting enzymes, facilitates glycoprotein folding (for reviews, see Refs. 2 and 3). In a widely accepted model (4), the partial deglucosylation of asparagine-linked Glc 3 Man 9 -GlcNAc 2 induces cotranslational physical interaction between glycoproteins and members of a small family of lectins, each of which recognizes the monoglucosylated glycan as ligand (2). Dissociation of the complex coincides with the removal of glucose by glucosidase II (5). In the absence of conformational maturation, UDP-glucose:glycoprotein glucosyltransferase (UGTR) functions as a folding sensor (6) that recognizes structural determinants common to nonnative glycoprotein structure (7,8). Reglucosylation of asparagine-linked oligosaccharides induces the reassembly of folding intermediates with calnexin (4). As such, reversible glucosylation hinders premature exit from the ER (2, 4, 9) until correctly folded molecules that are no longer substrates for UGTR are released from the lectin-mediated retention cycle (4).As a rule, failure to attain conformational maturation following biosynthesis results in the selective elimination of misfolded polypeptides and unassembled protein complexes by a relatively stringent mechanism of conformation-based quality control (10, 11). Molecular characterization of primary and secondary disposal systems, plus the identification of the full reperto...

In eukaryotes, proteins destined for secretion are translocated as nascent polypeptides into the lumen of the endoplasmic reticulum (ER) 1 (for a review, see Ref. 1). Folding into the native conformation, a structure dictated by the primary amino acid sequence (2), is facilitated through transient interaction with one or more molecular chaperones (3). Conformational fidelity of folded structures is monitored by a poorly understood quality control system (4) which prevents transport of incompletely folded and unassembled proteins beyond the ER (5).Cotranslational addition of Glc 3 Man 9 GlcNAc 2 to specific asparagine residues and hydrolysis of attached glucose units can accompany translocation of the nascent polypeptide (6). Reglucosylation of high mannose-type glycans has been detected in microsomal preparations from mammals, plants, fungi, yeast, and protozoa (7,8) and is catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (UGTR) (8 -10). Importantly, only high mannose-type oligosaccharides attached to unfolded proteins function as acceptors in the glucose transfer reaction (11-13). Results from a cell-free system indicate that the unfolded polypeptide and asparagine-linked GlcNAc are responsible for eliciting glucose transfer (13).Several nascent proteins (14 -18) form transient associations with calnexin (also designated p88 or IP90), a calcium-binding protein of the ER membrane (19). Since calnexin functions as a molecular chaperone for glycoproteins (17,20) and interacts with monoglucosylated oligosaccharides (21), Hammond et al. (15) proposed that reglucosylation by UGTR may function to initiate assembly between unfolded glycoproteins and the molecular chaperone. In support of this idea, Labriola et al. (22) have reported that delivery of a nascent acid hydrolase to lysosomes of Trypanosoma cruzi is delayed by inhibition of ER ␣-glucosidase activity and is a predictable response if attached monoglucosylated oligosaccharides are interacting with calnexin.Recent evidence suggests that protein folding and quality control machinery may participate in the molecular pathogenesis of several human diseases caused by defective intracellular transport of an aberrantly folded protein through the secretory pathway (23-25). Human ␣ 1 -antitrypsin (AAT) is a 394-amino acid protein (26, 27) glycosylated at three specific asparagine residues (28). It is folded into a highly ordered tertiary structure containing three ␤-sheets, nine ␣ helices, and three internal salt bridges (29). The human AAT structural gene is highly polymorphic (30), and several alleles exhibit a distinct mutation predicted to preclude conformational maturation of the encoded polypeptide following biosynthesis (31). Secretion of AAT from hepatocytes (32, 33) is impaired in response to incomplete folding of the polypeptide (34,35).AAT is a member of the serine proteinase inhibitor superfamily (36). Since elastase released by activated neutrophils is rendered inactive by the inhibitor (37), diminished circulating levels...