Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast. Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides. Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified. The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase. Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio. Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of [3H]Glc3[14C]Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic analysis of the products showed that [3H]Glc was removed only in the presence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor.
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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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