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Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex.

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of ≈500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-Å resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation. [ABSTRACT FROM AUTHOR]

Studies the mechanism of protein N-glycosylation. Characteristics of the asparagine-linked glycosylation protein; Origin of the concept that oligosaccharyl transferase (OT) functions as an enzyme; Mechanistic complexities of OT; Role of amino acids adjacent to consensus sequence in the binding of polypeptide substrate with OT complex.

Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide-diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cells with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose-labeled or [3H]glucosamine-labeled oligosaccharide-lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP-Man. Furthermore, during the first 5 min after the addition of cycloheximide to LM cells, when [3H]mannose incorporation into oligosaccharide-lipid was maximally reduced, the specific activity of the GDP-Man pool was identical to that observed in control cells. Labeling in vivo of the lipid-linked saccharide precursors of oligosaccharide-lipid revealed that cycloheximide had virtually no effect on the synthesis of Man-P-Dol, GlcNAc-PP-Dol, GlcNAc2-PP-Dol, and β-Man-(GlcNAc)2-PP-Dol (Dol = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid-linked saccharide precursors. Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 µg/ml) resulted in a 300% stimulation of oligosaccharide-lipid synthesis when compared to non-supplemented cells. However, dolichyl phosphate supplementation of cycloheximide-treated cells failed to restore oligosaccharide-lipid synthesis to the level observed in control cells. In control cells pre-labeled oligosaccharide-lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. Howeve... [ABSTRACT FROM AUTHOR]

Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbp1p may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations m the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 °C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex. [ABSTRACT FROM AUTHOR]