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.
Henquet
Maurice
M
Laboratory of Plant Physiology, Wageningen University, 6703 BD Wageningen, The Netherlands.
Lehle
Ludwig
L
Schreuder
Mariëlle
M
Rouwendal
Gerard
G
Molthoff
Jos
J
Helsper
Johannes
J
van der Krol
Sander
S
Bosch
Dirk
D
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