Oligosaccharyltransferase (OTase) glycosylates selected asparagine residues in secreted and membrane proteins in eukaryotes, and asparagine (N)-glycosylation affects the folding, stability and function of diverse glycoproteins. The range of acceptor protein substrates that are efficiently glycosylated depends on the action of several accessory subunits of OTase, including in yeast the homologous proteins Ost3p and Ost6p. A model of Ost3p and Ost6p function has been proposed in which their thioredoxin-like active site cysteines form transient mixed disulfide bonds with cysteines in substrate proteins to enhance the glycosylation of nearby asparagine residues. We tested aspects of this model with a series of in vitro assays. We developed a whole protein mixed disulfide interaction assay that showed that Ost6p could form mixed disulfide bonds with selected cysteines in pre-reduced yeast Gas1p, a model glycoprotein substrate of Ost3p and Ost6p. A complementary peptide affinity chromatography assay for mixed disulfide bond formation showed that Ost3p could also form mixed disulfide bonds with cysteines in selected reduced tryptic peptides from Gas1p. Together, these assays showed that the thioredoxin-like active sites of Ost3p and Ost6p could form transient mixed disulfide bonds with cysteines in a model substrate glycoprotein, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase in vivo.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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