title: Arabidopsis thaliana ALG3 mutant synthesizes immature oligosaccharides in the ER and accumulates unique N-glycans.
abstract: The core oligosaccharide Glc(3)Man(9)GlcNAc(2) is assembled by a series of membrane-bound glycosyltransferases as the lipid carrier dolichylpyrophosphate-linked glycan in the endoplasmic reticulum (ER). The first step of this assembly pathway on the ER luminal side is mediated by ALG3 (asparagine-linked glycosylation 3), which is a highly conserved reaction among eukaryotic cells. Complementary genetics compared with Saccharomyces cerevisiae ALG gene families and bioinformatic approaches have enabled the identification of ALG3 from other species. In Arabidopsis thaliana, AtALG3 (At2g47760) was identified as alpha1,3-mannosyltransferase. Complementation analysis showed that AtALG3 rescued the temperature-sensitive phenotype, that lipid-linked oligosaccharide assemblies and that protein underglycosylation of S. cerevisiae ALG3-deficient mutant. In Arabidopsis ALG3 mutant, an immature lipid-linked oligosaccharide structure, M5(ER), was synthesized, and used for protein N-glycosylation, resulting in the blockade of subsequent maturation with the concanavalin A affinoactive and Endo H-insensitive structure. N-Glycan profiling of total proteins from alg3 mutants exhibited a unique structural profile, alg3 has rare N-glycan structures including Man(3)GlcNAc(2), M4(ER), M5(ER) and GlcM5(ER), which are not usually detected in Arabidopsis, and a much less amount of complex-type N-glycan than that in wild type. Interestingly, despite protein N-glycosylation differences compared with wild type, alg3 showed no obvious phenotype under normal and high temperature or salt/osmotic stress conditions. These results indicate that AtALG3 is a critical factor for mature N-glycosylation of proteins, but not essential for cell viability and growth in Arabidopsis.
authors.affiliation: The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Osaka 565, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kajiura
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Seki
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D009844:Oligosaccharides; D008364:Mannosyltransferases; C121563:alpha 1,3-mannosyltransferase