Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-α-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 theNterminus, 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 a- xylosidase, whereas no reaction was catalyzed by b- 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 onlyknownin rhamnogalacturonan- II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan- II isolated from bothRGXT1andRGXT2T- DNAinsertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2enhanced 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 dyeBODIPYTRceramide, 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.