Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexin͞ calreticulin, CNX͞CRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. T he folding status of glycoproteins is strictly censored in the endoplasmic reticulum (ER), before they attain their final destination to membranes, organelles, or the extracellular environment (1). Proteins that fail to fold properly are initially retained in the ER and eventually degraded in the proteasomes. The quality control system involves the action of UDPGlc:glycoprotein glucosyltranferase (GT) and glucosidase II (GII) enzymes and the ER resident lectins calnexin (CNX) and calreticulin (CRT). Proteins that are incompletely folded are glucosylated by GT, thus generating Glc 1 Man 9 GlcNAc 2 structures. On binding those structures, CNX and͞or CNR retain the monoglucosylated glycoproteins in the ER. Eventually, the Glc units added by GT are removed by GII, causing the release of glycoproteins from the lectin anchors. Glucosylationdeglucosylation cycles catalyzed by the opposing activities of GT and GII continue until native conformations are attained. Glycoproteins then become substrates of GII but not of GT and are thus free to pursue their travel through the secretory pathway.The CNX͞CRT-monoglucosylated glycan interaction is one of the mechanism by which cells retain incompletely folded glycoproteins in the ER and, in addition, it enhances folding efficiency by preventing protein aggregation and allowing intervention of additional ER chaperones and folding accessory proteins. The key element in this mechanism is GT as it is the only component exclusively acting on incompletely folded glycoproteins. This feature is based on the recognition of two structural determinants not exposed in native conformations. One of them is the innermost GlcNAc unit, which is buried within the protein scaffold in native but solvent exposed in nonnative conformations (2). In addition, there is an ill-defined determinant in incompletely folded polypeptides that is also recognized by GT. This protein determinant has eluded identification so far, mainly because of the difficulties intrinsic to the study of partly folded conformations of possible intermediates along the folding pathway of nascent polypeptides. It has been suggested that hydrophobic residues exposed in incompletely folded conformers could be the protein elements recognized by GT as exp...

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 (GCI2-(1-63)V63S and GCI2-(1-64)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.A quality control mechanism in the endoplasmic reticulum (ER) 1 is in charge of sensing the folding state of glycoproteins before their transport to the Golgi apparatus (1). Proteins incorrectly folded are initially retained in the ER and eventually degraded by the proteasome, a process known as ER-associated degradation (2, 3). Recent findings on glycoprotein processing in the ER showed that sugar moieties could be exploited to encode information on glycoprotein folding status. About 65% of Asn-XSer/Thr consensus sequences in proteins entering the ER lumen are N-glycosylated (4). The Glc 3 Man 9 GlcNAc 2 glycan transferred is deglucosylated immediately by the sequential action of glucosidases I and II. At this stage the proteins that are not correctly folded are reglucosylated by UDP-Glc:glycoprotein glucosyltransferase (GT), generating Glc 1 Man 9 GlcNAc 2 . This enzyme has a unique property as it glucosylates glycoproteins displaying native-close, molten globule-like but not random coil or compactnative conformations. Monoglucosylated glycans are specifically recognized by two ER resident lectins, calnexin (CNX) and calreticulin (CRT), in charge of retaining folding intermediates and irreparably misfolded glycoproteins in the ER. In addition, lectin binding facilitates conformational maturation of glycoproteins by preventing aggregation and allowing the labor of lectin-associated proteins such as ERp57 (endoplasmic reticulum protein 57), a protein of the protein disulfide isomerase family (5).The single gluc...

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Members of the Rhizobiaceae family synthesize cyclic ␤-(1,2)-glucans through a mechanism which involves oligosaccharides covalently linked to a large inner membrane protein.Upon elongation to a polymer of about 15 to 25 glucose units, the oligosaccharides are cycled and thus liberated from the protein anchor. The glucose acceptor role of the inner membrane protein and the transient character of its glucosylation have been clearly demonstrated in Agrobacterium tumefaciens and Rhizobium meliloti (35), Rhizobium fredii (4, 5), Rhizobium loti (19), and all biovars of Rhizobium leguminosarum (9). After neutral cyclic ␤-(1,2)-glucans are formed, some of them are substituted by phosphoglycerol and/or succinyl residues, probably inside the periplasmic space (3,6,13,20,21,32).The A. tumefaciens chv and R. meliloti ndv chromosomal regions code for the protein intermediates ChvB and NdvB, respectively, of approximately 319 kDa (15,33). In addition, these regions code for the ChvA/NdvA protein, which is probably involved in the transport of ␤-(1,2)-glucans to the periplasmic space (16,23,28).It is likely that formation of cyclic ␤-(1,2)-glucan requires at least the following three enzymatic activities: (i) one that catalyzes the transfer of the first glucose to an unknown amino acid residue in the protein intermediate, (ii) a glucosyltransferase activity responsible for chain elongation, and (iii) an activity responsible for glucan cyclization and release from the protein. Due to the fact that only cyclic glucan forms have been detected after release from the protein intermediate (34), cyclization and release reactions may proceed in the same reaction step (31,36). In this paper, we present evidence indicating that a unique protein component carries all three activities. We also suggest that this protein component is likely to be the protein intermediate. MATERIALS AND METHODSBacterial strains and culture media. A. tumefaciens and R. meliloti strains (Table 1) were grown in TY medium (0.5% tryptone and 0.3% yeast extract) and yeast extract-mannitol medium (1% mannitol, 0.1% yeast extract, 0.05% K 2 HPO 4 , 0.02% MgSO 4 and 0.02% NaCl), respectively. Bacteria were grown at 28ЊC in a rotary shaker.Inner membrane preparation. Inner membranes were purified by fractional centrifugation as previously described (24) and resuspended in 30 mM Tris-HCl buffer, pH 8.2.Native polyacrylamide gel electrophoresis. Native polyacrylamide gel electrophoresis (PAGE) was carried out in running gels of different acrylamide content (3, 5, or 7%) with an acrylamide/bisacrylamide ratio of 30:0.8, in 0.2 M Tris-HCl (pH 8.8)-0.1% Triton X-100. Gels were polymerized 20 h before electrophoresis in order to inactivate free radicals generated during polymerization. Agarose (0.7%) was added to the 3% polyacrylamide gel to improve manipulation. The stacking gels contained in all cases 3.5% acrylamide-0.1% Triton X-100-0.1 M Tris-HCl (pH 6.8) and were polymerized with 5 g of riboflavine per ml, TE-MED (N,N,NЈ,NЈ-tetramethylethylenediamine), and irradiatio...

It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP-glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER-containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP-glucose was temperature dependent and saturable with an apparent Km of 46 microM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP-N-acetylglucosamine did not enter these vesicles. Demonstration of UDP-glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP-glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Delta, lack cell wall beta-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Delta mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.