Oligosaccharyl transferase: gatekeeper to the secretory pathway

Dempski, Robert E.; Imperiali, Barbara

doi:10.1016/s1367-5931(02)00390-3

Cited by 112 publications

(78 citation statements)

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“…In addition, in the current study Ost1p was found to be crosslinked to Alg1p, which raises the possibility of interaction of the lipid-linked oligosaccharide assembly pathway and the Nglycosylation process. In fact, enzymes involved in protein translocation followed by N-glycosylation, protein folding, and formation of disulfide bonds in the ER may be tightly coupled (2). Thus it is possible that OT is not only in proximity to the Sss1p component of the Sec61 complex, but also to the enzymes of the lipid-linked oligosaccharide synthesis pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, this idea was further supported by the cross-linking of OT subunit Ost1p Ost1p with another ER membrane protein, Alg1p, with regard to their relationship as follows: several OT subunits (Ost1p, Wbp1p, Swp1p) were found containing the consensus sequence, which is distinctive among dolichol binding enzymes in their membrane-spanning domains and has been proposed to bind to the assembled lipid-linked oligosaccharides (25,26), and Alg1p was recently found to exist as a homo-oligomer and perform dual functions in sugar transfer and in the recognition of dolichol phosphate-derived substrates. 2 Therefore, Alg1p could serve as a good model to investigate our hypothesis if it ideally interacts with one or more OT subunits that are involved in recognition and the binding of lipid-linked oligosaccharide donors. As expected, our result (as shown in Fig.…”

Section: Selectivity Of the Cross-linker-as Demonstrated Inmentioning

confidence: 99%

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

Yan

Ahmed

Qi³

et al. 2003

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Selectivity Of the Cross-linker-as Demonstrated Inmentioning

confidence: 99%

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

Yan

Ahmed

Qi³

et al. 2003

Journal of Biological Chemistry

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“…N-glycosylation is a predominant modification of proteins in eukaryotic organisms 1–3 . N-linked glycans serve many essential functions, affecting protein folding and sorting in the endoplasmic reticulum (ER) and mediating interactions of the cell or organism with its environment 4 .…”

Section: Introductionmentioning

confidence: 99%

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Bai

Wang

Zhao

et al. 2018

Nature

103

131

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SUMMARY N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalyzed by an eight-protein oligosaccharyltransferase complex, OST, embedded in the ER membrane. Our understanding of eukaryotic protein N-glycosylation has been limited due to the lack of high-resolution structures. Here we report a 3.5-Å resolution cryo-EM structure of the Saccharomyces cerevisiae OST, revealing the structures of Ost1–5, Stt3, Wbp1, and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N-glycan mediates interaction with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST-Sec61 translocon interface, funneling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides novel insights into co-translational protein N-glycosylation and may facilitate the development of small-molecule inhibitors targeting this process.

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“…The ER houses the machinery for asparagine-linked glycosylation as well as for disulfide formation (10,11). Proteins in the secretory pathway that are misfold in the ER (12,13) are subject to retrograde transport into the cytosol, where they are degraded by proteasomes (14,15).…”

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confidence: 99%

The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment

Bosques

Imperiali

2003

Proc. Natl. Acad. Sci. U.S.A.

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It is now accepted that the structural transition from cellular prion protein (PrP C ) to proteinase K-resistant prion protein scrapie (PrP Sc ) is the major event leading to transmissible spongiform encephalopathies. Although the mechanism of this transition remains elusive, glycosylation has been proposed to impede the PrP C to PrP Sc conversion. To address the role of glycosylation, we have prepared glycosylated and unglycosylated peptides derived from the 175-195 fragment of the human prion protein. Comparison of the structure, aggregation kinetics, fibril formation capabilities, and redox susceptibility of Cys-179 has shown that the N-linked glycan (at Asn-181) significantly reduces the rate of fibrillization by promoting intermolecular disulfide formation via Cys-179. Furthermore, the aggressive fibrillization of a C179S mutant of this fragment highlights the significant role of disulfide stability in retarding the rate of fibril formation. The implications of these studies are discussed in the context of fibril formation in the intact prion protein.S pongiform encephalopathies are a group of fatal neurodegenerative diseases that can be manifested sporadically, genetically inherited, or in some cases transmitted (1-3). In contrast to many diseases where the nature of the infectious particle is virus or bacterium, currently, the only agent associated with these conditions is the structural isoform of the cellular prion protein (PrP C ) known as the scrapie conformation (PrP Sc ) (1, 4). PrP C is an N-linked glycoprotein that is normally attached to the cell membrane by a glycosylphosphatidylinositol anchor (5). PrP C also contains an intramolecular disulfide bond (Cys179OCys-214) that provides structural stability to the C terminus of the protein (6, 7). Secondary structure analyses have shown that PrP C is a predominantly helical protein, whereas PrP Sc is mainly ␤-sheet, suggesting that the conversion into PrP Sc involves a major structural change (8). Although the structure of the unglycosylated PrP C monomer has been solved by NMR spectroscopy (7, 9), elucidation of the PrP Sc structure has been hampered by its highly aggregated state.As with many membrane-associated proteins, initiation of the biosynthesis of PrP begins with translocation into the endoplasmic reticulum (ER) where the amino-terminal signal sequence is cleaved (1). The ER houses the machinery for asparagine-linked glycosylation as well as for disulfide formation (10, 11). Proteins in the secretory pathway that are misfold in the ER (12, 13) are subject to retrograde transport into the cytosol, where they are degraded by proteasomes (14, 15). In particular, protein isoforms (or mutants) that are less stable during their maturation are most likely to undergo this retrograde transport (14). Lindquist and coworkers have recently shown that inhibition of the proteasome causes PrP to accumulate in the cytosol, where it can adopt a PrP Sc -like conformation (16,17). Furthermore, expression of an unglycosylated, cytosolic form of PrP has extreme...

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Oligosaccharyl transferase: gatekeeper to the secretory pathway

Cited by 112 publications

References 50 publications

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

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

The atomic structure of a eukaryotic oligosaccharyltransferase complex

The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment

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