The essential yeast <i>NLT1</i> gene encodes the 64 kDa glycoprotein subunit of the oligosaccharyl transferase

Pathak, Rahul; Parker, Carl S.; Imperiali, Barbara

doi:10.1016/0014-5793(95)00253-6

Cited by 25 publications

(15 citation statements)

References 27 publications

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“…Previous biochemical investigations of the yeast OST suggested that the enzyme consists of four (Ost1p, Wbp1p, Ost3p, Swp1p) (21,23), five (68), or six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p, Ost5p) (22). Genetic screens have identified in addition STT3 (35) and OST4 (34).…”

Section: Ost3 and Ost6 Are Required For N-glycosylation In Vivo And mentioning

confidence: 99%

“…Meanwhile, however, OST complexes have been purified from different sources, such as dog pancreas (19,20), yeast (21)(22)(23), hen oviduct (24), and human (25) and pig (26) liver. The subunit composition of the various isolated complexes and the protein sequences, so far obtained, reveal in part a high conservation of the structural organization of this enzyme throughout evolution.…”

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confidence: 99%

“…Independent OST purifications from Saccharomyces cerevisiae have yielded active complexes consisting of four polypeptides (Ost1p (64/62 kDa), Wbp1p (47 kDa), Ost3p (34 kDa), and Swp1p (30 kDa)) (21,23) or of six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p (16 kDa), and Ost5p (9.5 kDa)) (22). Cloning and functional analysis of OST1 (23,27), WBP1 (28), SWP1 (29), and OST2 (30) have indicated that these genes are essential for the vegetative growth of the yeast cell and reveal significant homology to components of the canine complex: to ribophorin I (27), OST48 (31), to the C-terminal half of ribophorin II (22), and DAD1 (defender against apoptotic cell death) (20), respectively. In contrast, OST3 (32) and OST5 (33) coding for the 34-and 9.5-kDa subunits, respectively, are not essential, but their deletion yields glycosylation defects and reduces OST activity in vitro.…”

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confidence: 99%

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The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

Knauer

Lehle

1999

Journal of Biological Chemistry

112

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The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an ⌬ost3⌬ost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a ϳ240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.

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Section: Ost3 and Ost6 Are Required For N-glycosylation In Vivo And mentioning

confidence: 99%

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confidence: 99%

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The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

Knauer

Lehle

1999

Journal of Biological Chemistry

112

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“…T he enzyme catalyzing N-linked glycosylation, called oligosaccharyl transferase (OT), is a remarkably complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, is composed of nine different transmembrane proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Even though genes encoding the nine subunits of yeast OT have been cloned, at present there is very limited information about the function of each of the subunits.…”

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confidence: 99%

Studies on the function of oligosaccharyl transferase subunits: A glycosylatable photoprobe binds to the luminal domain of Ost1p

Qi¹,

Lennarz²

2002

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

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Oligosaccharyl transferase (OT) is a complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, contains nine different transmembrane proteins. One of our goals is to identify the OT subunit(s) responsible for recognizing the consensus sequence, -Asn-X-Thr͞Ser-, and catalyzing the oligosaccharide transfer reaction. By using a substrate-based photoprobe, earlier we found that Ost1p was specifically linked to the radiolabeled photoprobe. We have now examined Ost1p in more detail. Deletion of the cytoplasmic tail of Ost1p caused no defects in growth and glycosylation. In addition, replacement of the transmembrane domain with other hydrophobic amino acids did not impair growth. In contrast, a construct containing only the luminal domain of Ost1p did not support cell growth. Given these observations, we concentrated on studying the luminal domain of Ost1p and localized the photoprobe attachment region within a sequence of nine amino acid residues. Because mutations in the photoprobe attachment region did not cause any severe growth or glycosylation defects, we conclude that this region is not involved in the recognition of the N-glycosylation site. By further mutagenesis of the conserved residues of Ost1p we conclude that the luminal domain mediates interactions with other subunits of OT and becomes labeled because of its proximity to the recognition and͞or catalytic subunit in the OT complex, Stt3p.T he enzyme catalyzing N-linked glycosylation, called oligosaccharyl transferase (OT), is a remarkably complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, is composed of nine different transmembrane proteins (1-16). Even though genes encoding the nine subunits of yeast OT have been cloned, at present there is very limited information about the function of each of the subunits. We have undertaken the study of the mode of interaction of these transmembrane protein subunits within the endoplasmic reticulum to elucidate their function.To identify the subunit(s) of yeast OT that recognizes -Asn-X-Thr͞Ser-sites that can be glycosylated (17-19), we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125 Ilabeled acceptor tripeptide, 125 I-Bolton-Hunter (bh)-Asn-BpaThr-Amide. By using this 125 I-labeled Bpa containing tripeptide, which was shown to be a substrate of OT, we found that after photoactivation of yeast microsomes, Ost1p was specifically labeled (20).Our current objective was to localize the site of binding of the photoprobe and to determine whether Ost1p is the actual subunit that recognizes the N-glycosylation consensus sequence -Asn-X-Thr͞Ser-. Three models to explain probe attachment to Ost1p were considered (Fig. 1). In model I, interaction of the Asn and Thr residues of the photolabeled tripeptide with the putative N-glycosylation site recognition domain occurs, and the photoreactive middle amino acid, Bpa, covalently binds to some nearby amino acid residue in the Ost1p backbone. Alternatively, in mod...

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“…Biochemical, molecular biological, and genetic studies have led to the identification of a remarkably large number of subunits for yeast OT (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). During the last decade, OT complexes have been purified from different sources, such as dog pancreas (21,22), hen oviduct (23), human (24) and pig liver (25), and yeast (10,26,27).…”

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Studies on the Function of Oligosaccharyl Transferase Subunits

Qi¹,

Lennarz

2002

Journal of Biological Chemistry

145

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In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.

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The essential yeast NLT1 gene encodes the 64 kDa glycoprotein subunit of the oligosaccharyl transferase

Cited by 25 publications

References 27 publications

The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae

Studies on the function of oligosaccharyl transferase subunits: A glycosylatable photoprobe binds to the luminal domain of Ost1p

Studies on the Function of Oligosaccharyl Transferase Subunits

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