A Dual Affinity Tag on the 64-kDa Nlt1p Subunit Allows the Rapid Characterization of Mutant Yeast Oligosaccharyl Transferase Complexes

Pathak, Rahul; Imperiali, Barbara

doi:10.1006/abbi.1996.9812

Cited by 25 publications

(13 citation statements)

References 23 publications

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“…A similar composition was then reported for the avian, human, and pig OT complexes (6)(7)(8)31). In early studies, the purification of the yeast enzyme complex was carried out, which indicated that the complex might be composed of four to six subunits (18,30,34,35). In a later study, all subunits were reported to be present in the purified OT complex based on their migration on SDS͞ PAGE gels (27).…”

Section: Resultssupporting

confidence: 61%

“…The OT complex has been purified from various mammalian and yeast sources by using a combination of chromatographic techniques (3-8, 18, 27, 30, 31, 33, 34), and other studies have used the immunoaffinity technique (13,27,34,35). The purification of the OT complex was first carried out from canine pancreatic microsomes as a complex of three polypeptides: ribophorin I, ribophorin II, and OST48 (mammalian homologs of Ost1p, Swp1p, and Wbp1p, respectively) (3, 33).…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

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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“…Complex by Specific Interactions with Each Other The concept that OT functions as an enzyme complex originated from the earlier biochemical studies where it was demonstrated that in a variety of organisms multiple components were detected in the purified fraction that exhibited highest specific OT activity (6,8,9,(12)(13)(14). Thus far, it is not certain how these subunits interact to form a catalytically active complex in vivo.…”

Section: The Nine-yeast Ot Subunits Form a Catalytically Activementioning

confidence: 99%

Unraveling the Mechanism of Protein N-Glycosylation

Yan

Lennarz

2005

Journal of Biological Chemistry

190

119

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“…Previous biochemical studies of the yeast oligosaccharyltransferase suggested that the enzyme contains four, five, or six subunits (3)(4)(5)31). Here we have utilized yeast strains that express epitope-tagged OST subunits to define the in vivo subunit composition and stoichiometry of the enzyme.…”

Section: In Vivo Analysis Of the Subunit Composition Of The Ost-mentioning

confidence: 99%

The Highly Conserved Stt3 Protein Is a Subunit of the Yeast Oligosaccharyltransferase and Forms a Subcomplex with Ost3p and Ost4p

Karaoglu

Kelleher

Gilmore

1997

Journal of Biological Chemistry

109

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The oligosaccharyltransferase has been purified from Saccharomyces cerevisiae as an hetero-oligomeric complex composed of four or six subunits. Here, the in vivo subunit composition and stoichiometry of the oligosaccharyltransferase were investigated by attaching an epitope coding sequence to a previously characterized subunit gene, OST3. Five (Ost1p, Wbp1p, Swp1p, Ost2p, and Ost5p) of the seven polypeptides that were coimmunoprecipitated with the epitope-tagged Ost3p were identical to those obtained by the conventional purification procedure. Two additional coprecipitating polypeptides with apparent molecular masses of 60 and 3.6 kDa were identified as the 78-kDa Stt3 protein and the 36-residue Ost4 protein, respectively. Stt3p and Ost4p were previously identified in screens for gene products involved in N-linked glycosylation. Quantification of the in vivo radiolabeled subunits and the radioiodinated purified enzyme shows that the yeast oligosaccharyltransferase is composed of equimolar amounts of eight subunits. Exposure of the immunoprecipitated oligosaccharyltransferase to mild protein denaturants yielded a subcomplex comprised of Stt3p, Ost3p, and Ost4p. These experiments, taken together with genetic and biochemical evidence for subunit interactions, suggest that the enzyme is composed of the following three subcomplexes: (a) Stt3p-Ost4p-Ost3p, (b) Swp1p-Wbp1p-Ost2p, and (c) Ost1p-Ost5p.N-Glycosylation of proteins is an essential, highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST) 1 catalyzes the transfer of a preassembled high mannose oligosaccharide (Glc 3 Man 9 GlcNAc 2 ) onto Asn-X-Ser/Thr acceptor sites on nascent polypeptides as they are translocated into the lumen of the rough endoplasmic reticulum (for reviews see Refs. 1, and 2). Biochemical, molecular biological, and genetic studies have led to the identification of a surprisingly large number of proteins that are required for the expression of wild-type OST activity.The yeast OST was initially purified as an oligomeric complex composed of six subunits that are designated as Ost1p (62/64 kDa), Wbp1p (45 kDa), Ost3p (34 kDa), Swp1p (30 kDa), Ost2p (16 kDa), and Ost5p (9 kDa) (3). However, catalytically active tetrameric OST complexes that appear to lack Ost2p and Ost5p have also been described (4, 5). In addition, the 34-kDa Ost3 protein appears to be present in reduced amounts relative to the other three subunits in the purified OST tetramer (4), raising the possibility that oligomeric forms of the OST may exist that differ with respect to the presence of regulatory or accessory subunits.Prior to purification of the yeast enzyme, genetic and biochemical studies had established that Wbp1p and Swp1p were essential for in vivo and in vitro expression of OST activity (6, 7). Mutations in genes encoding Ost1p, Ost2p, Ost3p, and Ost5p also cause substantial reductions in both the N-linked glycosylation of proteins in vivo and the transfer of dolichollinked oligosaccharides to ac...

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A Dual Affinity Tag on the 64-kDa Nlt1p Subunit Allows the Rapid Characterization of Mutant Yeast Oligosaccharyl Transferase Complexes

Cited by 25 publications

References 23 publications

Dimeric organization of the yeast oligosaccharyl transferase complex

Dimeric organization of the yeast oligosaccharyl transferase complex

Unraveling the Mechanism of Protein N-Glycosylation

The Highly Conserved Stt3 Protein Is a Subunit of the Yeast Oligosaccharyltransferase and Forms a Subcomplex with Ost3p and Ost4p

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