Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane.

Abstract

The mechanism of translocation in vitro of sugar nucleotides and adenosine 3-phosphate 5'-phosphosulfate (PAPS) into the lumen of rat liver Golgi apparatus vesicles has been studied. It has been previously shown that the Golgi apparatus membrane has specific carrier proteins for PAPS and sugar nucleotides. We now report that translocation of the above nucleotide derivatives across Golgi membranes occurs via a coupled equimolar exchange with the corresponding nucleoside monophosphates. An initial incubation of Golgi vesicles with GDP-fucose radiolabeled in the guanidine ring resulted in accumulation within the lumen of radiolabeled GMP. Exit of GMP from these vesicles was specifically dependent on the entry of (additional) GDP-fucose into the vesicles (GDPmannose and other sugar nucleotides had no effect). GDP-fucose-stimulated exit of GMP was temperature dependent, was blocked by inhibitors of GDP-fucose transport, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and appeared to be equimolar with GDP-fucose entry. Preliminary evidence for specific, equimolar exchange of CMP-N-acetylneuraminic acid with CMP, PAPS with 3'-AMP, and UDP-galactose and UDP-N-acetylglucosamine with UMP was also obtained. These results strongly suggest the existence of different antiport proteins within the Golgi membrane that mediate the 1:1 exchange of sugar nucleotides or PAPS with the corresponding nucleoside monophosphate. Such proteins may have a regulatory role in glycosylation and sulfation reactions within the Golgi apparatus.Recent studies from this laboratory have shown that rat liver Golgi-derived vesicles can translocate in vitro CMP-N-acetylneuraminic acid (AcNeu), GDP-fucose, UDP-N-acetylglucosamine (GIcNAc), and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) from an external compartment into a lumenal one (1-5). These reactions were found to be (i) saturable at high concentrations of sugar nucleotides and PAPS, (ii) temperature dependent, (iii) inhibited by treatment of the Golgi vesicles with proteases under conditions where lumenal marker enzymes were not inhibited, and (iv) inhibited competitively by the corresponding nucleoside mono-, di-, and triphosphate (6). Since the above sugar nucleotides and PAPS did not inhibit translocation of each other, it was hypothesized that there were different translocator proteins in the membrane of the Golgi apparatus and that portions of these proteins face the cytoplasmic side of the Golgi apparatus membranes. Evidence for translocation of UDP-galactose (Gal) into Golgi vesicles from mammary gland and rat liver (7,8), CMP-AcNeu into rat liver Golgi (9) and hen oviduct microsomes (10) has also been obtained in other laboratories.The aim of the present study was to understand the energy mechanism by which the above sugar nucleotides and PAPS are translocated across the Golgi vesicle membranes. We now present evidence suggesting that such a mechanism involves exchange with the corresponding nucleoside monophosphate via an antiport protein.

The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP‐glucose into the endoplasmic reticulum, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana

“…To confirm this result we performed the UDPglucose transport assays in liposomes reconstituted in the presence of microsomal AtUTr3-containing membranes. Figure 2a shows that import of UDP-glucose into liposomes reconstituted with AtUTr3 was dependent on preloading the liposomes with uridine 5¢-phosphate (UMP), indicating that AtUTr3 functions as a UDP-glucose/UMP antiporter, a feature that has been previously shown for other nucleotide sugar transporters (Capasso and Hirschberg, 1984;Rollwitz et al, 2006). In order to further characterize the biochemical features of the transporter, the kinetic constants of AtUTr3 were assessed.…”

“…The concentration of UDP-glucose in the cytosol of plant cells is in the millimolar range. On the other hand, the K m for the nucleotide sugars of nucleotide sugar transporters is in the micromolar range (Capasso and Hirschberg, 1984;Waldman and Rudnick, 1990;Rollwitz et al, 2006) and in the case of AtUTr3 the K m for UDP-glucose is below 10 lM, therefore under normal circumstances it is likely that the transport of UDP-glucose is occurring at saturating concentrations and at V max . Thus, a higher demand for UDP-glucose in the lumen of the ER would require an increased number of UDP-glucose transporters at the ER membrane.…”

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

“…Evidence from mammalian cells indicates that GDP-Fuc is transported into the Golgi lumen by a transporter protein located in the membrane (Capasso and Hirschberg, 1984a). To determine whether the uptake of GDP-Fuc into pea stem Golgi vesicles is also mediated by a putative membrane protein, we analyzed the protease sensitivity of GDP-Fuc uptake into Golgi vesicles.…”

Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice

“…The Arabidopsis genome contains more than 40 NSTs (7). Such abundance correlates with the diversity of carbohydrates and the substrate specificity of individual NSTs that generally act as antiporters (8). The relationship between sequence identity and substrate specificity is weak, probably because the sequence motifs required for substrate recognition in NSTs are not well defined.…”