Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants.
N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.
Strasser
Richard
R
Institute of Applied Genetics and Cell Biology, Vienna, Austria. richard.strasser@boku.ac.at
Schoberer
Jennifer
J
Jin
Chunsheng
C
Glössl
Josef
J
Mach
Lukas
L
Steinkellner
Herta
H
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D006020:Glycopeptides; D006056:Golgi Apparatus / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D025942:Oligosaccharides, Branched-Chain / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection
D006020:Glycopeptides; D025942:Oligosaccharides, Branched-Chain; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase