GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter

Abstract

The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan ␣,1-2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls.

The Import of S-Adenosylmethionine into the Golgi Apparatus Is Required for the Methylation of Homogalacturonan

“…1B). XG-FT is a type II membrane protein with a catalytic site that faces the lumen of the Golgi apparatus (Perrin et al, 1999;Wulff et al, 2000). Since HGA-MT and XG-FT were measured in the presence of acceptors that cannot cross the membrane, the increases we detected in their activity following the permeabilization of the Golgi vesicle membranes could be explained by the luminal localization of the active site of both enzymes.…”

“…We thus performed our SAM import assay in the presence of DIDS, a general inhibitor of the NSTs (Wulff et al, 2000). Under conditions where DIDS produced a 10% to 15% decrease in the uptake of SAM, the incorporation of GDP-Fuc was found to be decreased by 70% (Fig.…”

“…To determine the mechanisms underlying the importation of SAM into the Golgi, we purified Golgi vesicles from etiolated pea epicotyls, an experimental model plant that has been utilized previously to analyze the topology of enzymes involved in the biosynthesis of polysaccharides, as well as the transport of metabolites and ions (Muñ oz et al, 1996;Neckelmann and Orellana, 1998;Wulff et al, 2000;Sterling et al, 2001;Ordenes et al, 2002). We first determined the orientation of the catalytic domain of HGA-MT in our Golgi vesicle preparations by measuring its enzymatic activity in the presence of exogenous HGA as the acceptor for the methylation reaction.…”

Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator-Homologous Transporters, Classified by Their Putative Substrate-Binding Site

“…In plants, nucleotide sugars are mainly used in the synthesis of noncellulose polysaccharides and glycoproteins of the cell wall that takes place in the lumen of the Golgi cisternae (Dupree and Sherrier, 1998). Because the majority of NSTs are specific for only one nucleotide sugar, several transporters should be necessary for the transport of substrates for the syntheses of pectin and hemicellulose (Wulf et al, 2000).…”

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

“…GTs transfer the sugar residue from an activated nucleotide donor, in the form of a UDP-or GDPsugar, to a growing polysaccharide chain. Most GTs are type-II membrane-bound proteins with a catalytic domain facing the Golgi lumen (Sterling et al, 2001;Wulff et al, 2000;Scheible and Pauly, 2004). However, most nucleotide sugars utilized by GTs are produced in the cytosol (Bar-Peled and O'Neill, 2011;Bonin et al, 1997;Seifert, 2004).…”