Identification of the gene encoding the alpha1,3-mannosyltransferase (ALG3) in Arabidopsis and characterization of downstream n-glycan processing.
Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man5GlcNAc2-PP-Dol alpha1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Deltaalg3 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 Arabidopsis 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.
Laboratory of Plant Physiology, Wageningen University, 6703 BD Wageningen, The Netherlands.
Maurice
M
Henquet
Ludwig
L
Lehle
Mariëlle
M
Schreuder
Gerard
G
Rouwendal
Jos
J
Molthoff
Johannes
J
Helsper
Sander
S
van der Krol
Dirk
D
Bosch
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005816:Genetic Complementation Test; D006031:Glycosylation; D015151:Immunoblotting; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D011134:Polysaccharides; C036483:T-DNA; D008364:Mannosyltransferases