During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc(3)Man(9)GlcNAc(2)-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and glycosylphosphatidylinositol anchor assembly. In support of this conclusion, mutant cells expressing UPPS (1) synthesized (55)Dol-P based on MS analysis, (2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo, and (3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [(3)H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the endoplasmic reticulum of Chinese hamster ovary cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [(14)C](55)polyprenyl-P-P intermediate when incubated with [(14)C]isopentenyl pyrophosphate and [(3)H]Man-P-(55)Dol when incubated with GDP-[(3)H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the endoplasmic reticulum of yeast and mammalian cells, and that conserved sequences in the cis-isoprenyltransferases are recognized by, yet to be identified, binding partners in the endoplasmic reticulum of mammalian cells.
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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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