Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo

In higher eukaryotes a quality control system monitoring the folding stateof glycoproteins is located in the ER and is composed of the proteinscalnexin, calreticulin, glucosidase II, and UDP-glucose: glycoproteinglucosyltransferase. It is believed that the innermost glucose residue ofthe N- linked oligosaccharide of a glycoprotein serves as a tag in thiscontrol system and therefore performs an important function in the proteinfolding pathway. To address this function, we constructed Saccharomycescerevisiae strains which contain nonglucosylated (G0), monoglucosylated(G1), or diglucosylated (G2) glycoproteins in the ER and used these strainsto study the role of glucose residues in the ER processing ofglycoproteins. These alterations of the oligosaccharide structure did notresult in a growth phenotype, but the induction of the unfolded proteinresponse upon treatment with DTT was much higher in G0 and G2 strains ascompared to wild-type and G1 strains. Our results provide in vivo evidencethat the G1 oligosaccharide is an active oligosaccharide structure in theER glycoprotein processing pathway of S.cerevisiae. Furthermore, byanalyzing N- linked oligosaccharides of the constructed strains we candirectly show that no general glycoprotein glucosyltransferase exists in S.cerevisiae.