Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol.
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 GlcNAc(2)-PP-dolichol (Dol), the second step in the biosynthesis of the unique lipid-linked core oligosaccharide. Down-regulation of each gene led to a defect in protein N-glycosylation and an accumulation of GlcNAc(1)-PP-Dol in vivo as revealed by metabolic labeling with [(3)H]glucosamine. Microsomal membranes from cells repressed for ALG13 or ALG14, as well as detergent-solubilized extracts thereof, were unable to catalyze the transfer of N-acetylglucosamine from UDP-GlcNAc to [(14)C]GlcNAc(1)-PP-Dol, but did not impair the formation of GlcNAc(1)-PP-Dol or GlcNAc-GPI. Immunoprecipitating Alg13p from solubilized extracts resulted in the formation of GlcNAc(2)-PP-Dol but required Alg14p for activity, because an Alg13p immunoprecipitate obtained from cells in which ALG14 was down-regulated lacked this activity. In Western blot analysis it was demonstrated that Alg13p, for which no well defined transmembrane segment has been 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 GlcNAc(2)-PP-Dol.
Bickel
Tanja
T
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
Lehle
Ludwig
L
Schwarz
Markus
M
Aebi
Markus
M
Jakob
Claude A
CA
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D015536:Down-Regulation; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008861:Microsomes / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000502:physiology*; D009844:Oligosaccharides / Q000737:chemistry*; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
D003902:Detergents; D007074:Immunoglobulin G; D008055:Lipids; D009844:Oligosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C016418:dolichyl-diphosphate-di-N-acetylchitobiose; C506082:Alg13 protein, S cerevisiae; C506083:Alg14 protein, S cerevisiae; D017351:N-Acetylglucosaminyltransferases