Biosynthesis of arabinogalactan-protein in Lolium multiflorum (Italian ryegrass) endosperm cells. Subcellular distribution of galactosyltransferases

Abstract

Intracellular membranes from protoplasts of Italian-ryegrass (Lolium multiflorum) endosperm cells have been fractionated on sucrose density gradients and identified on the basis of putative-marker-enzyme assays. Galactosyltransferases capable of incorporating galactose from UDP galactose into 66% ethanol-soluble products are associated with all membrane fractions. Affinity chromatography of the ethanol-insoluble products on (murine myeloma protein J539)-Sepharose reveals that the enzymes responsible for the synthesis of polymers containing (1----6)-beta-D-galactose residues are associated exclusively with subcellular fractions enriched in Golgi-derived membranes. This suggests that the Golgi apparatus plays an important part in the synthesis of the carbohydrate component of the ryegrass arabinogalactan-protein.

Identification and Characterization of in Vitro Galactosyltransferase Activities Involved in Arabinogalactan-Protein Glycosylation in Tobacco and Arabidopsis

“…Cloning and tagging of the Hyp:GalT and Gal:GalT enzymes in the future should allow for more precise subcellular localizations. Moreover, the subcellular localization of AGP GalT activities in ryegrass (Mascara and Fincher, 1982;Schibeci et al, 1984) and radish root (Kato et al, 2003), AGP fucosyltransferase activity in radish root (Misawa et al, 1996), an AGP fucosyltransferase enzyme in tobacco leaves (Wu et al, 2010), and AGP arabinosyltransferase activity in tobacco cultured cells (Kawasaki, 1987) likewise are consistent with involvement of the endomembrane system, particularly the Golgi apparatus, in AG biosynthesis.…”

“…Karr (1972) partially characterized a microsomal fraction that arabinosylated extensin peptides, and Bolwell (1986) suggested a lipid-linked intermediate might be involved in HRGP glycosylation. In addition, galactosyltransferase (GalT) activity associated with AGPs was identified in ryegrass (Lolium multiflorum; Mascara and Fincher, 1982) and pea (Pisum sativum; Hayashi and Maclachlan, 1984) membrane preparations; moreover, this activity in ryegrass was restricted to Golgi-derived membranes (Schibeci et al, 1984). Others identified an a-fucosyltransferase activity (Misawa et al, 1996) and a b-glucuronyltransferase activity (Endo et al, 2003) that might be involved in biosynthesis of radish (Raphanus sativus) root AGPs.…”

Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes of plant cells

“…These authors also reported that JIM13 reacts on Western blots with secreted material (prepared from BY-2 tobacco cell suspensions) with a molecular mass between 40 and 180 kDa, which is quite similar to our observation with maize roots.The localization of AGPs in the Golgi apparatus is also supported by biochemical evidence. For example, both precursors of AGPs (Kawasaki et al 1981(Kawasaki et al , 1987 and glycosyl transferases, which can participate on AGP biosynthesis (e.g., Schibeci et al 1984, Misawa et al 1996, were found in Golgi membrane fractions. Here, we show that another AGP epitope containing GlcA (as recognized by LM2 antibody) is present throughout the entire Golgi apparatus including its cis-, medial-, and trans-cisternae as well as the TGN of both monocotyledonous and dicotyledonous species represented by maize and sundew.…”

Identification of a novel group of putative Arabidopsis thaliana β-(1,3)-galactosyltransferases

“…In bacteria, at least 15 GalTs have been characterised and unlike their eukaryotic counterparts, they do not share the same topology: they can be either membranebound or soluble (Hennet 2002). In plant cells, membranebound GalTs are involved in the biosynthesis of pectins, galactomannans, xyloglucans and AGPs (McNab et al 1968;Panayotatos and Villemez 1973;Goubet et al 1993;Goubet and Morvan 1994;Geshi et al 2000;Peugnet et al 2001;Mascara and Fincher 1982;Schibeci et al 1984;Edwards et al 1999) as well as plastid galactolipids (Awai et al 2001).…”