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

Guangtao Li,Qi Yan,Aleksandra Nita-Lazar,Robert S Haltiwanger,William J Lennarz

doi:10.1074/jbc.m410969200

Abstract

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(539)NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn(539) is essential for its function in the OT complex.

Highlights

Oligosaccharyl transferase (OT)1 catalyzes the N-glycosylation of most secretory and membrane-bound proteins
Based on the TMHMM program, this site is located in the ER lumen and is a potential N-glycosylation site
It is known that three of the essential subunits, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins in the yeast OT complex, but no information has been available regarding whether the N-glycosylation of these three subunits is important for OT activity

Summary

Introduction

Oligosaccharyl transferase (OT) catalyzes the N-glycosylation of most secretory and membrane-bound proteins. Stt3p has been shown to be involved in cell wall ␤-1,6-glycan biosynthesis in S. cerevisiae and to interact with protein kinase cascade components via its N-terminal domain, which is oriented toward the cytosol. This function may be independent of its role in the recognition process of substrates, which involves the C-terminal domain that is oriented toward the lumen [5, 6]. We have addressed this question by site-directed mutation of potential N-glycosylation sites on the Ost1p, Wbp1p, and Stt3p subunits. Because our knowledge of the minimal spacing required for efficient N-glycosylation at two adjacent sites was limited, we addressed this question in the current study

Methods

Results

Conclusion