Carbohydrate modification of secreted, luminal, and integral membrane proteins is an essential eukaryotic cellular process. Folding, stability, localization, and recognition are some of the many properties contributed to glycoproteins by oligosaccharides. Oligosaccharyl transferase catalyzes the covalent attachment of oligosaccharides to asparagine residues within selected Asn-Xaa-Ser/Thr sequences of polypeptides translocated into the endoplasmic reticulum. Oligosaccharyl transferase was purified from a solubilized fraction of Saccharomyces cerevisiae membrane proteins by a sequence of chromatography on concanavalin A-agarose, heparin-agarose, and Qsepharose. Enzymatic activity copurified with an apparent complex of four polypeptides with molecular weights of 64 kDa, 45 kDa, 34 kDa, and 30 kDa. The 45 kDa glycoprotein and the 30 kDa protein were identified as the previously described proteins Wbplp and Swp1p. The essential NLT1 gene encoding the 64 kDa glycoprotein subunit of the enzyme was cloned using obtained amino acid sequences. The NLT1 gene encodes a 476 amino acid polypeptide with a signal sequence, a carboxyterminal hydrophobic domain, and four potential glycosylation sites. Antiserum prepared from a six-histidine tagged version of the NLT1 gene product expressed in E.coli recognized the two glycoforms of the 64 kDa protein in a crude yeast lysate and in the purified native enzyme complex. To further the characterization of the multisubunit enzyme, a version of Nlt1p with carboxy-terminal FLAG epitope and six-histidine tags was expressed from the CUP1 promoter. The construct expressing the affinity-tagged Nlt1p complemented a deletion of the NLT1 locus yielding oligosaccharyl transferase activity levels indistinguishable from strains expressing the native enzyme. Affinity purification of the tagged enzyme yielded a multimeric complex with polypeptide subunits of 70 kDa, 45 kDa, 34 kDa, 30 kDa, and 16 kDa; these subunits have been identified as the products of the tagged NLTI gene and the native WBP1, OST3, SWPI, and OST2 genes. The purified affinity-tagged oligosaccharyl transferase has been used to examine the minimum polypeptide composition necessary for catalytic activity and to probe the enzyme's peptide binding site with a series of photoaffinity labels.
Rahul Pathak (Tue,) studied this question.