New Findings on Interactions among the Yeast Oligosaccharyl Transferase Subunits Using a Chemical Cross-linker

Abstract

At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of crosslinking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.Over the past decade it has been established that in both higher eukaryotes and yeast, the enzyme oligosaccharyl transferase (OT), 1 is a complex consisting of multiple, nonidentical membrane protein subunits residing in the endoplasmic reticulum (ER) (1). In the case of the yeast Saccharomyces cerevisiae, nine different subunits have been cloned and identified. Among them, the genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; OST4 gene is essential for growth of the cell at 37°C, but not at 25°C; Ost3p, Ost5p, Ost6p subunits are not essential for the viability of the cell, but are required for maximal activity of the enzyme complex in catalyzing N-glycosylation (for review, see Refs. 1 and 2). To understand how these nine subunits interact with each other, a genetic approach using the yeast two-hybrid screen was utilized, but it failed to show interactions between any of the luminal domains of the yeast OT subunits (3). Studies on the mechanism of enzyme catalysis have proposed that Wbp1p may contain a site for the binding of the lipidlinked oligosaccharide substrate (4), and Stt3p was shown to be directly involved in peptide substrate recognition and/or the catalytic glycosylation process (5). However, there is little detailed information on how these two subunits interact to catalyze the glycosylation reaction.Study of the structural interactions of membrane proteins of the ER offers a special challenge because of the hydrophobicity of the proteins and the special environ...

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…3a–b). This architecture is consistent with a previous crosslinking studies 33 . The interfaces among the three subcomplexes in the membrane region are loose, with only a few protein-protein interactions between Ost2 and Stt3.…”

Dimeric organization of the yeast oligosaccharyl transferase complex

“…Because we have antibodies to all of the OT subunits except Ost4p, Ost3p, and Ost6p, we generated a yeast strain in which genes encoding Ost4p, Ost3p, and Ost6p were epitope-tagged. We chose to epitope tag the C terminus of these three subunits because it has been previously demonstrated that the C-terminal tagging of these subunits does not lead to any growth defect (22,26,28,29). We tagged the chromosomal copies of these three subunits and did not express them on an overexpression plasmid, thereby eliminating the possibility of artifactual complex formation.…”

“…Recently, it has been reported that the luminal domain of Wbp1p may possess the divalent metal ion-binding site (20), which has been shown to be essential for the catalysis of the OT reaction (21). Because Ost1p has been found to cross-link with a number of proteins, irrespective of their glycosylation status (22)(23)(24), it is proposed that the luminal domain of Ost1p may be involved in funneling the newly synthesized polypeptides into the active site of the OT reaction (24). However, it is important to note that with the exception of Stt3p, the contributions of the other OT subunits in the N-glycosylation reaction are highly speculative and based on indirect evidence.…”

“…However, it is important to note that with the exception of Stt3p, the contributions of the other OT subunits in the N-glycosylation reaction are highly speculative and based on indirect evidence. A possible conformational arrangement of the yeast OT complex has been proposed based on studies using a chemical cross-linker and the split-ubiquitin two-hybrid technique (22,25). Other researchers have proposed the presence of three subcomplexes within the OT complex (26).…”

Unraveling the Mechanism of Protein N-Glycosylation

“…However, recently application of a cross-linker, dithiobissuccinimidylpropionate, which bears a spacer arm of 12 Å, to study the interrelationship of any two of all of the yeast OT subunits has yielded a model that is not completely consistent with the above "subcomplex" model (18). In this study, we found that Ost3p and Ost6p cannot co-immunoprecipitate with each other suggesting that they do not exist in the same complex, but they showed exactly the same interaction pattern with all other OT subunits.…”