Sulfhydryl Modification of the Yeast Wbp1p Inhibits Oligosaccharyl Transferase Activity

Pathak, Rahul; Hendrickson, Tamara L.; Imperiali, Barbara

doi:10.1021/bi00013a005

Cited by 56 publications

(62 citation statements)

References 22 publications

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“…Kinetic models that are based on in vitro studies predict that substrate selection for fully glucosylated LLOs is accomplished by the OST complex via an allosteric regulation involving a regulatory and a catalytic binding site for the LLO substrate (Pathak et al 1995;Karaoglu et al 2001;Kelleher et al 2007). Studies performed with mammalian OSTs led to insights into possible functions of other complex subunits.…”

Section: Functions Of Ost Subunitsmentioning

confidence: 99%

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

Breitling

Aebi

2013

Cold Spring Harbor Perspectives in Biology

244

204

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The attachment of glycans to asparagine residues of proteins is an abundant and highly conserved essential modification in eukaryotes. The N-glycosylation process includes two principal phases: the assembly of a lipid-linked oligosaccharide (LLO) and the transfer of the oligosaccharide to selected asparagine residues of polypeptide chains. Biosynthesis of the LLO takes place at both sides of the endoplasmic reticulum (ER) membrane and it involves a series of specific glycosyltransferases that catalyze the assembly of the branched oligosaccharide in a highly defined way. Oligosaccharyltransferase (OST) selects the Asn-X-Ser/Thr consensus sequence on polypeptide chains and generates the N-glycosidic linkage between the side-chain amide of asparagine and the oligosaccharide. This ER-localized pathway results in a systemic modification of the proteome, the basis for the Golgi-catalyzed modification of the N-linked glycans, generating the large diversity of N-glycoproteome in eukaryotic cells. This article focuses on the processes in the ER. Based on the highly conserved nature of this pathway we concentrate on the mechanisms in the eukaryotic model organism Saccharomyces cerevisiae.

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Section: Functions Of Ost Subunitsmentioning

confidence: 99%

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

Breitling

Aebi

2013

Cold Spring Harbor Perspectives in Biology

244

204

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“…The structures of Ost4 were solved by NMR 19,20 . Biochemical studies suggested that Ost1 and Wbp1 recognize acceptor and donor substrates, respectively 8,21,22 . The structures of the eukaryotic OST have been limited to low-resolution EM reconstructions, hindering a mechanistic understanding of protein N-glycosylation in eukaryotes 23–26 .…”

Section: Introductionmentioning

confidence: 99%

“…These results suggest that both Wbp1 and Swp1 are involved in recruiting LLO. Indeed, Wbp1 and its mammalian homolog OST48 were shown to crosslink with LLO 21,39 .…”

Section: Introductionmentioning

confidence: 99%

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Bai

Wang

Zhao

et al. 2018

Nature

102

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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“…Ost4p is proposed to be responsible for recruiting Ost3p or Ost6p into the OT complex (16). Other studies have indicated that the donor substrate may be recognized by Wbp1p (17)(18)(19). 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).…”

mentioning

confidence: 99%

Dimeric organization of the yeast oligosaccharyl transferase complex

Chavan

Chen

et al. 2006

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

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The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of Ϸ500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-Å resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.electron microscopy ͉ membrane protein purification ͉ protein N-glycosylation

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Sulfhydryl Modification of the Yeast Wbp1p Inhibits Oligosaccharyl Transferase Activity

Cited by 56 publications

References 22 publications

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Dimeric organization of the yeast oligosaccharyl transferase complex

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