Structural basis for two-step glucose trimming by glucosidase II involved in ER glycoprotein quality control (original) (raw)
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Conformational Requirements for Glycoprotein Reglucosylation in the Endoplasmic Reticulum
The Journal of Cell Biology, 2000
Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted b...
Quaternary and Domain Structure of Glycoprotein Processing Glucosidase II
Biochemistry, 2001
Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, R and. The R subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between R and subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (R 1 1) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the R 1 1 complex was confirmed by its high susceptibility to proteases. The subunit could be proteolytically removed from the R 1 1 complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the R subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the R subunit, which can function independently of the subunit, while the complete R and subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the subunit may retain the complete R 1 1 complex in the ER.
The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stagefolding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V max /K m ) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.
UDP-GlC:glycoprotein glucosyltransferase-glucosidase II, the ying-yang of the ER quality control
Seminars in Cell & Developmental Biology, 2010
The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectinassociated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.
A sweet code for glycoprotein folding
FEBS Letters, 2015
Glycoprotein synthesis is initiated in the endoplasmic reticulum (ER) lumen upon transfer of a glycan (Glc3Man9GlcNAc2) from a lipid derivative to Asn residues (N‐glycosylation). N‐Glycan‐dependent quality control of glycoprotein folding in the ER prevents exit to Golgi of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin‐chaperones (calnexin and calreticulin) that recognize monoglucosylated polymannose protein‐linked glycans, lectin‐associated oxidoreductase acting on monoglucosylated glycoproteins (ERp57), a glucosyltransferase that creates monoglucosylated epitopes in protein‐linked glycans (UGGT) and a glucosidase (GII) that removes the glucose units added by UGGT. This last enzyme is the only mechanism component sensing glycoprotein...