N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Here we identify and characterize two essential yeast proteins having homology to bacterial glycosyltransferases, designated Alg13p and Alg14p, as being required for the formation of GlcNAc2-PP-Dol, the second step in the biosynthesis of the unique lipid-linked core oligosaccharide. Down-regulation of each gene leads to a defect in protein N-glycosylation and an accumulation of GlcNAc1-PP-Dol in vivo as revealed by metabolic labeling with [3H]glucosamine. Microsomal membranes from cells repressed for ALG13 or ALG14, as well as detergent-solubilized extracts thereof, are unable to catalyze the transfer of N-acetylglucosamine from UDP-GlcNAc to [14C]GlcNAc1-PP-Dol, but do not impair the formation of GlcNAc1-PP-Dol or GlcNAc-GPI. Immunoprecipitating Alg13p from solubilized extracts results in formation of GlcNAc2-PP-Dol, but requires Alg14p for activity, since an Alg13p immunoprecipitate obtained from cells, in which ALG14 was down regulated, lacks this activity. In Western blot analysis it is demonstrated that Alg13p, for which no well defined transmembrane segment is predicted, localizes both to the membrane and cytosol, the latter form, however, is enzymatically inactive. In contrast, Alg14p is exclusively membrane-bound. Repression of the ALG14 gene causes a depletion of Alg13p from the membrane. By affinity chromatography on IgG-Sepharose using Alg14-ZZ as bait, we demonstrate that Alg13-myc co-fractionates with Alg14-ZZ. The data suggest that Alg13p associates with Alg14p to a complex forming the active transferase catalyzing the biosynthesis of GlcNAc2-PP-Dol.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|