Transport of UDP-galactose in Plants

Abstract

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-sideout Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgienriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1-MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDPgalactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.

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

“…Reagents that react selectively with the ⑀-amino group of Lys residues like pyridoxal-5Ј-phosphate (PLP), 2,4,6-trinitrobenzene sulfonate, or DIDS are strong inhibitors of phosphate translocator activities (Flü gge and Heldt, 1986;Rumpho et al, 1988;Gross et al, 1990). DIDS also inhibits the activity of the UDP-Gal transporter from Arabidopsis (Norambuena et al, 2002). Both PLP and DIDS were shown to react with the same Lys group of the TPT and PPT (Gross et al, 1990).…”

“…Thus, it is reasonable to assume that these proteins are also homodimers that mediate an antiport transport of so far unknown substrates. (d) 4,4Ј-diisothiocyanstilbene-2,2Ј-disulfonic acid (DIDS), an inhibitor of the pPT activity (Flü gge and Heldt, 1986), also leads to a reduction of the transport activity of NST proteins (Norambuena et al, 2002).…”

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

“…In Arabidopsis thaliana, the genes encoding for NSTs are similar to those encoding for plastidic triose phosphate translocators (TPTs); together, 44 NSTs and 7 TPTs form a gene family of 51 members (Knappe et al, 2003;Rautengarten et al, 2014). To date, a number of these NSTs from Arabidopsis have been functionally characterized, specifically transporters for GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), UDP-rhamnose (UDP-Rha), and UDP-xylose (UDP-Xyl) (Bakker et al, 2005;Baldwin et al, 2001;Ebert et al, 2015;Handford et al, 2012Handford et al, , 2004Norambuena et al, 2002Norambuena et al, , 2005Rautengarten et al, 2014Rautengarten et al, , 2016Rollwitz et al, 2006).…”

Characterization of AtNST‐KT1, a novel UDP‐galactose transporter from Arabidopsis thaliana

“…Only nine of the 43 NST proteins in Arabidopsis have been analyzed in detail so far, namely members of the GONST family, GONST1 to GONST4, that transport GDPMan [9,18] and two members of a family that was named UTR according to the AtUTr proteins which have been described by Norambuena and colleagues [23,24]. AtUTr1 was the first NST from plants shown to transport UDP-activated sugars, namely UDP-Gal and UDP-Glc [23] while a second transporter, AtUTr2, was shown to solely transport UDPGal [24]. Three NST families have been named NST-KD, NST-KT and NST-KVAG according to a specific dipeptide that is part of the putative substrate binding site [11].…”

“…First, mutants of yeast or other organisms that are defective in a particular nucleotide sugar transport activity were functionally complemented by (heterologous) NSTs thereby allowing the characterization of the corresponding substrate specificities [9,17,18,23,25]. Second, most of the NSTs have been characterized by measuring the transport of radiolabelled nucleotide sugars into microsomes or Golgi enriched vesicles isolated from rat liver or yeast that overexpress particular NST proteins [9,10,17,23,24]. However, characterization of NSTs in transport assays using Golgi vesicles could be complicated by a high background of endogenous transport activities and by competing glycosyltransferases that also use nucleotide sugars as substrates [26].…”