Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II.
Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-alpha-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-alpha-d-xylose to l-fucose. The disaccharide product was hydrolyzed by alpha-xylosidase, whereas no reaction was catalyzed by beta-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an alpha-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-alpha-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A-sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.
Egelund
Jack
J
Biotechnology Group, Danish Institute of Agricultural Sciences and Center for Molecular Plant Physiology, DK-1871 Frederiksberg C, Denmark.
Petersen
Bent Larsen
BL
Motawia
Mohammed Saddik
MS
Damager
Iben
I
Faik
Ahmed
A
Olsen
Carl Erik
CE
Ishii
Tadashi
T
Clausen
Henrik
H
Ulvskov
Peter
P
Geshi
Naomi
N
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D006056:Golgi Apparatus / Q000201:enzymology*; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010368:Pectins / Q000096:biosynthesis*; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000097608:UDP Xylose-Protein Xylosyltransferase
D029681:Arabidopsis Proteins; D017931:DNA Primers; D007527:Isoenzymes; D010368:Pectins; D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase; C042492:rhamnogalacturonan II