Die2 (also called Alg10) is a multi-pass ER membrane glucosyltransferase of the ALG10 family (GT-C superfamily). It catalyses the final step of lipid-linked oligosaccharide assembly: it transfers glucose from dolichyl-phosphate-glucose in Ξ±1,2 linkage onto Glc2Man9GlcNAc2-PP-dolichol to give the complete Glc3Man9GlcNAc2-PP-dolichol donor (EC 2.4.1.256). The reaction takes place on the lumenal side of the ER membrane. The terminal Ξ±1,2-glucose is a key recognition element for the oligosaccharyltransferase, so only fully assembled LLO is used efficiently. DIE2 is not essential. alg10 mutants accumulate Glc2Man9GlcNAc2-PP-dolichol and hypoglycosylate secreted proteins, and they show synthetic growth defects with reduced OST activity (wbp1). DIE2 was first isolated as a gene that, in multicopy, raises expression of the inositol transporter gene ITR1 and of INO1, although its deletion does not abolish ITR1 expression and the link to its glycosylation activity is unknown.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004583 dolichyl-phosphate-glucose-glycolipid alpha-glucosyltransferase activity | TAS Reactome:R-SCE-449661 | MODIFY | Summary: dolichyl-phosphate-glucose-glycolipid alpha-glucosyltransferase activity, TAS from Reactome R-SCE-449661 ('Addition of a third glucose to the N-glycan skeleton by Alg10'). Correct but general. A specific child term (GO:0106073) exists for the terminal Ξ±1,2-glucosylation step. Reason: Make specific with the reaction-level term. Proposed replacements: dolichyl pyrophosphate Glc2Man9GlcNAc2 alpha-1,2-glucosyltransferase activity Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
| GO:0005783 endoplasmic reticulum | HDA PMID:26928762 One library to make them all: streamlining the creation of y... | ACCEPT | Summary: endoplasmic reticulum, HDA from the SWAp-Tag GFP library (Yofe et al. 2016). Consistent with Die2 being an ER membrane enzyme. Supporting Evidence: PMID:26928762 we found that most new localization assignments were to the ER, punctate structures and vacuole |
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: is_active_in endoplasmic reticulum, IBA from PANTHER PTN000312712 (ALG10 family; Arabidopsis, rat, human and yeast donors). Correct. Supporting Evidence: PMID:9597543 The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: ER membrane, IEA from the UniProt subcellular-location mapping (SL-0097). Correct: UniProt predicts 13 transmembrane segments. Supporting Evidence: file:yeast/DIE2/DIE2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | IMP PMID:9597543 The ALG10 locus of Saccharomyces cerevisiae encodes the alph... | ACCEPT | Summary: ER membrane, IMP from Burda & Aebi 1998. alg10 mutants have an LLO-glucosylation defect in the ER, and Die2 is an ER membrane glucosyltransferase. Correct. Supporting Evidence: PMID:9597543 The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-SCE-449661 | ACCEPT | Summary: ER membrane, TAS from Reactome R-SCE-449661. Correct. Supporting Evidence: PMID:9597543 The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. |
| GO:0005829 cytosol | RCA GO_REF:0000123 | REMOVE | Summary: is_active_in cytosol, RCA from the YeastPathways LLO pathway compartment. The compartment is wrong for Die2/Alg10. Die2 uses Dol-P-Glc to add the terminal glucose to Glc2Man9GlcNAc2-PP-Dol, which is the last lumenal LLO step. Reason: Pathway-level compartment over-propagation. The Die2 reaction is lumenal. Supporting Evidence: PMID:32103179 The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates |
| GO:0006487 protein N-linked glycosylation | IBA GO_REF:0000033 | ACCEPT | Summary: protein N-linked glycosylation, IBA from PANTHER PTN000312712. Correct. The terminal glucose is needed for efficient recognition of the LLO by OST. Supporting Evidence: PMID:9597543 Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. |
| GO:0006487 protein N-linked glycosylation | IMP PMID:9597543 The ALG10 locus of Saccharomyces cerevisiae encodes the alph... | ACCEPT | Summary: protein N-linked glycosylation, IMP from Burda & Aebi 1998. alg10Ξ strains hypoglycosylate secreted proteins. Correct. Supporting Evidence: PMID:9597543 Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | IDA PMID:9597543 The ALG10 locus of Saccharomyces cerevisiae encodes the alph... | ACCEPT | Summary: dolichol-linked oligosaccharide biosynthetic process, IDA from Burda & Aebi 1998 (in vitro Dol-P-Glc-dependent glucosylation of Glc2Man9GlcNAc2-PP-Dol). Core process. Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: dolichol-linked oligosaccharide biosynthetic process, IEA from InterPro IPR016900 (Alg10). Correct. Supporting Evidence: PMID:9597543 we have identified alg10 mutant strains which accumulate lipid-linked Glc2Man9GlcNAc2 |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | RCA GO_REF:0000123 | ACCEPT | Summary: dolichol-linked oligosaccharide biosynthetic process, RCA from YeastPathways. Correct. Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | TAS PMID:10336995 Ordered assembly of the asymmetrically branched lipid-linked... | ACCEPT | Summary: dolichol-linked oligosaccharide biosynthetic process, TAS from Burda et al. 1999, a study of ordered LLO assembly by the ER glycosyltransferases. Correct. Supporting Evidence: PMID:10336995 The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. |
| GO:0071944 cell periphery | HDA PMID:26928762 One library to make them all: streamlining the creation of y... | MARK AS OVER ANNOTATED | Summary: cell periphery, HDA from the SWAp-Tag GFP library (Yofe et al. 2016), where 'cell periphery' is one of the visual categories. Die2 is a polytopic ER membrane enzyme. In yeast, cortical ER lies just under the plasma membrane and gives a peripheral signal, which most likely explains this call. No plasma-membrane or cell-surface function is known. Reason: Most likely reflects the cortical ER. The ER membrane location is already captured. Supporting Evidence: PMID:26928762 Assignments were: Ambiguous; Below threshold; Bud; Bud neck; Cell periphery; Cytosol; ER |
| GO:0106073 dolichyl pyrophosphate Glc2Man9GlcNAc2 alpha-1,2-glucosyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Dol-P-Glc:Glc2Man9GlcNAc2-PP-Dol Ξ±1,2-glucosyltransferase activity, IBA from PANTHER PTN000312712 (ALG10 family). Conserved and well placed. The target has its own IDA support. Core. Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
| GO:0106073 dolichyl pyrophosphate Glc2Man9GlcNAc2 alpha-1,2-glucosyltransferase activity | IDA PMID:9597543 The ALG10 locus of Saccharomyces cerevisiae encodes the alph... | ACCEPT | Summary: GO:0106073, IDA from Burda & Aebi 1998. A novel in vitro assay showed that Alg10p adds the terminal Ξ±1,2-glucose using Dol-P-Glc. This is the defining reference for the GO term. Core. Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
| GO:0106073 dolichyl pyrophosphate Glc2Man9GlcNAc2 alpha-1,2-glucosyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: GO:0106073, IEA from combined methods (InterPro IPR016900, RHEA:29543, EC 2.4.1.256). Correct. Supporting Evidence: file:yeast/DIE2/DIE2-uniprot.txt adds the third and last glucose residue from dolichyl phosphate glucose |
| GO:0106073 dolichyl pyrophosphate Glc2Man9GlcNAc2 alpha-1,2-glucosyltransferase activity | RCA GO_REF:0000123 | ACCEPT | Summary: GO:0106073, RCA from YeastPathways (Dol-P-Glc + Glc2Man9GlcNAc2-PP-Dol). Correct. Supporting Evidence: PMID:9597543 show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide |
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Download this section (compressed HTML)Q: Die2/Alg10 belongs to a GT-C family distinct from Alg6/Alg8. Does it use the same conserved-module catalytic aspartate as Alg6?
Experiment: Determine a Die2/Alg10 structure with a Dol-P-Glc analogue and test candidate lumenal acidic residues by mutagenesis in the in vitro glucosylation assay.
Dolichyl-phosphoglucose-dependent alpha-1,2-glucosyltransferase; located in the ER; functions in pathway that synthesizes the dolichol-linked oligosaccharide precursor for N-linked protein glycosylation; has a role in regulation of ITR1 and INO1; human homolog ALG10B can complement yeast die2 null mutant
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