Studies on the N-Glycosylation of the Subunits of Oligosaccharyl Transferase in Saccharomyces cerevisiae

作者：Guangtao Li, Qi Yan, Aleksandra Nita‐Lazar, Robert S. Haltiwanger, William J. Lennarz · 发表于：Journal of Biological Chemistry · 年份：2004 · DOI：10.1074/jbc.m410969200 · 被引用次数：31 · 研究领域：Glycosylation and Glycoproteins Research、Carbohydrate Chemistry and Synthesis、Galectins and Cancer Biology

In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N(535)NTWN(539)NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man(8)GlcNAc(2) is found linked only to Asn(539). Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N(535)NTWN(5...