Arabidopsis_alg3 - Identification of the Gene Encoding the...

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Unformatted text preview: Identification of the Gene Encoding the a 1,3-Mannosyltransferase (ALG3) in Arabidopsis and Characterization of Downstream N-Glycan Processing W Maurice Henquet, a Ludwig Lehle, b Marie ¨lle Schreuder, a Gerard Rouwendal, c Jos Molthoff, c Johannes Helsper, c Sander van der Krol, a,1 and Dirk Bosch c,d,1,2 a Laboratory of Plant Physiology, Wageningen University, 6703 BD Wageningen, The Netherlands b Lehrstuhl fu ¨ r Zellbiologie und Pflanzenphysiologie, Universita ¨ t Regensburg, 93053 Regensburg, Germany c Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, 6708 PB Wageningen, The Netherlands d Membrane Enzymology, Department of Chemistry, Utrecht University, 3584 CH Utrecht, The Netherlands Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a man- nosyltransferase gene from Arabidopsis thaliana , which is the functional homolog of the ALG3 (Dol-P-Man:Man 5 GlcNAc 2- PP-Dol a 1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a D alg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2 , was identified in Arabi- dopsis with residual levels of wild-type ALG3 , derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response. INTRODUCTION In eukaryotes, secreted proteins may be modified on specific Asn residues by sugars upon entry into the endoplasmic reticulum (ER) in a process called N-glycosylation. The initial addition of N-glycan structure is to aid the folding process of the protein, and subsequent modifications of the N-glycans in the ER have a signaling function in the protein folding quality control mecha- nism. In mammals, N-glycans on glycoproteins that arise after further processing play crucial roles in many biological pro- cesses, and their structure and biosynthesis have been well studied (Kornfeld and Kornfeld, 1985; Fiedler and Simons, 1995; Ohtsubo and Marth, 2006). Plants also possess N-glycosylation, and the core structure of N-glycans in plants is similar to that found in mammals, but far less is known about their biosynthe-...
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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Arabidopsis_alg3 - Identification of the Gene Encoding the...

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