ABSTRACT
Asparagine-linked glycosylation of proteins is an essential cotranslational and posttranslational protein modification in plants. The central step in this process is the transfer of a preassembled oligosaccharide to nascent proteins in the endoplasmic reticulum by the oligosaccharyltransferase (OST) complex. Despite the importance of the catalyzed reaction, the composition and the function of individual OST subunits are still ill defined in plants. Here, we report the function of the highly conserved OST subunit OST3/6. We have identified a mutant in the OST3/6 gene that causes overall underglycosylation of proteins and affects the biogenesis of the receptor kinase EF-TU RECEPTOR involved in innate immunity and the endo-ß-1,4-glucanase KORRIGAN1 required for cellulose biosynthesis. Notably, the ost3/6 mutation does not affect mutant variants of the receptor kinase BRASSINOSTEROID-INSENSITIVE1. OST3/6 deficiency results in activation of the unfolded protein response and causes hypersensitivity to salt/osmotic stress and to the glycosylation inhibitor tunicamycin. Consistent with its role in protein glycosylation, OST3/6 resides in the endoplasmic reticulum and interacts with other subunits of the OST complex. Together, our findings reveal the importance of Arabidopsis (Arabidopsis thaliana) OST3/6 for the efficient glycosylation of specific glycoproteins involved in different physiological processes and shed light on the composition and function of the plant OST complex.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Hexosyltransferases/metabolism , Membrane Proteins/metabolism , Plant Immunity , Stress, Physiological , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cellulase/genetics , Cellulase/metabolism , Cellulose/metabolism , Endoplasmic Reticulum/metabolism , Glycoproteins , Glycosylation , Hexosyltransferases/genetics , Mannitol/pharmacology , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Protein Interaction Mapping , Protein Kinases/genetics , Protein Kinases/metabolism , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/metabolism , Recombinant Fusion Proteins , Seedlings/drug effects , Seedlings/enzymology , Seedlings/genetics , Seedlings/physiology , Sequence Alignment , Sodium Chloride/pharmacology , Tunicamycin/pharmacologyABSTRACT
Assembly of the dolichol-linked oligosaccharide precursor (Glc(3) Man(9) GlcNAc(2) ) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc(2) Man(9) GlcNAc(2) and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response.
