Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1

Abstract

Transport of nucleotide-sugars across the Golgi membrane is required for the lumenal synthesis of a variety of essential cell surface components, and is mediated by nucleotide sugar transporters (NSTs) which are members of the large drug/metabolite superfamily of transporters. Despite the importance of these proteins in plants, so far only two have been described, GONST1 and AtUTr1 from Arabidopsis thaliana. In this work, our aim was to identify further Golgi nucleotide-sugar transporters from Arabidopsis. On the basis of their sequence similarity to GONST1, we found four additional proteins, which we named GONST2, 3, 4 and 5. These putative NSTs were grouped into three clades: GONST2 with GONST1; GONST3 with GONST4; and GONST5 with six further uncharacterized proteins. Transient expression in tobacco cells of a member of each clade, fused to the Green Fluorescent Protein (GFP), suggested that all these putative NSTs are localised in the Golgi. To obtain evidence for nucleotide sugar transport activity, we expressed these proteins, together with the previously characterised GONST1, in a GDP-mannose transport-defective yeast mutant (vrg4-2). We tested the transformants for rescue of two phenotypes associated with this mutation: sensitivity to hygromycin B and reduced glycosylation of extracellular chitinase. GONST1 and GONST2 complemented both phenotypes, indicating that GONST2, like the previously characterized GONST1, is a GDP-mannose transporter. GONST3, 4 and 5 also rescued the antibiotic sensitivity, but not the chitinase glycosylation defect, suggesting that they can also transport GDP-mannose across the yeast Golgi membrane but with a lower efficiency. RT-PCR and analysis of Affymetrix data revealed partially overlapping patterns of expression of GONST1-5 in a variety of organs. Because of the differences in ability to rescue the vrg4 - 2 phenotype, and the different expression patterns in plant organs, we speculate that GONST1 and GONST2 are both GDP-mannose transporters, whereas GONST3, GONST4 and GONST5 may transport other nucleotide-sugars in planta.

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 recently, the first three proteins of these groups have been functionally characterized. One protein of the NST-KT family and one of the NST-KVAG family were shown to be UDP-Gal transporters [17] while a second protein of the NST-KVAG family might transport GDP-Man but also other nucleotide sugars [18]. These results and further extensive analysis of NST proteins revealed that the degree of sequence identity is not indicative for the substrate specificity of the transporters [6], i.e.…”

“…In Arabidopsis, 43 distinct genes encoding NSTs and seven genes (including one pseudogene) encoding pPTs could be identified while 43 NST genes and ten pPT genes were detected in rice. 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]. Two proteins belonging to the NST-KVAG family have been shown to transport UDP-Gal (AtUDP-GalT1) [17] and GDP-Man (GONST5) [18]. In contrast, the monospecific UDP-Gal transporter AtUDP-GalT2 [17] is a member of the NST-KT family.…”

Endoplasmic reticulum: Where nucleotide sugar transport meets cytokinin control mechanisms

“…SLC35D2 is phylogenetically related to UDP-Glc/UDP-Gal NST UTR7 17 from clade II, which also includes characterized and putative GDP-mannose transporters. 18,19 It remains to be tested whether any of the Arabidopsis NSTs from these 2 Blast similarity searches of available plant genomes revealed usually a single ROCK1 ortholog in other plant species ( Table 1) Table 1). No clear orthologous sequences were detected in available red algae genomes.…”