Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1? null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.     

The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.     

The role of nucleoside diphosphatase in a uridine nucleotide cycle associated with lactose synthesis in rat mammary-gland Golgi apparatus.

1. UDP-galactose utilization by isolated Golgi vesicles or rat mammary gland synthesizing lactose causes accumulation of UMP but not UDP, although UDP is the immediate product of lactose synthase (EC 2.4.1.22). 2. This can be ascribed to a nucleoside diphosphatase (EC 3.6.1.6), specific for UDP, GDP and IDP, activated by bivalent metal ions and apparently located on the luminal face of the Golgi membrane. 3. The uridine diphosphatase activity exceeds the total galactosyltransferase activity 5-fold, and is estimated to maintain UDP at about 14 micrometer within the Golgi lumen. 4. Evidence is given that UMP, but not UDP, penetrates the membrane and that UMP is rephosphorylated to UDP by a UMP kinase located in the cytosol. 5. Golgi-cytosol relationships with respect to lactose synthesis are formulated in terms of a uridine nucleotide cycle which throws new light on the energy cost and possible regulation of lactose synthesis.     

Nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments in Schizosaccharomyces pombe

Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1+ did not affect Golgi N- or O-glycosylation, whereas disruption of Spynd1+ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.     

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

Absence of nucleoside diphosphatase activities in the yeast secretory pathway does not abolish nucleotide sugar-dependent protein glycosylation

It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.     

Nucleotide sugar transporters: biological and functional aspects

The Golgi apparatus serves as the major site of glycosylation reactions. Nucleotide sugars which are substrates of the Golgi localized glycosyltransferases are synthesized in the cytoplasm (cell nucleus in case of CMP-sialic acid) and must be transported into the compartment lumen. This transport function is carried out by nucleotide sugar transporters. The first genes were cloned in the year 1996 and revealed a family of structurally conserved multi-transmembrane-spanning proteins. Due to the high structural and functional conservation, the identification of many putative nucleotide sugar transporter sequences has become possible in the existing gene data bases and accelerates the increase in knowledge on structure-function-relationships. Recent developments in the nucleotide sugar transporter field are discussed in this article.     

Membranfluß und Nucleosiddiphosphatase-Reaktion in Wurzelhaaren von Lepidium sativum

Summary Root hairs of Lepidium sativum were incubated with a Wachstein-Meisel medium in experiments designed to localize the activity of nucleoside diphosphatase(s). Electron dense precipitates were found in the ER and in Golgi cisternae of the secretory face of the dictyosomes and their adjacent Golgi vesicles. Such precipitates were absent in the Golgi cisternae of the regeneration face of the dictyosomes and in the detached Golgi vesicles which extrude pectic cell wall substances. These results may be the consequence of the normal cycle of membrane compounds associated with the secretion in which the nucleoside diphosphatase(s) participate (by activation and inactivation) as one of the cycling components. Alternatively the nucleoside diphosphatase(s) may undergo a special cycle in which they are transferred from one cisterna or its vesicles to the next as part of the process of cisternal maturation.     

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.     

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.     

Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum

The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999) FEBS Lett. 458, 309-312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-DeltaN proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default.     

Enzyme cytochemistry of the alveolar sacs and golgian-like cisternae in the ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trophozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Identification of a conserved motif in the yeast golgi GDP-mannose transporter required for binding to nucleotide sugar

Glycoproteins and lipids in the Golgi complex are modified by the addition of sugars. In the yeast Saccharomyces cerevisiae, these terminal Golgi carbohydrate modifications primarily involve mannose additions that utilize GDP-mannose as the substrate. The transport of GDP-mannose from its site of synthesis in the cytosol into the lumen of the Golgi is mediated by the VRG4 gene product, a nucleotide sugar transporter that is a member of a large family of related membrane proteins. Loss of VRG4 function leads to lethality, but several viable vrg4 mutants were isolated whose GDP-mannose transport activity was reduced but not obliterated. Mutations in these alleles mapped to a region of the Vrg4 protein that is highly conserved among other GDP-mannose transporters but not other types of nucleotide sugar transporters. Here, we present evidence that suggest an involvement of this region of the protein in binding GDP-mannose. Most of the mutations that were introduced within this conserved domain, spanning amino acids 280-291 of Vrg4p, lead to lethality, and none interfere with Vrg4 protein stability, localization, or dimer formation. The null phenotype of these mutant vrg4 alleles can be complemented by their overexpression. Vesicles prepared from vrg4 mutant strains were reduced in luminal GDP-mannose transport activity, but this effect could be suppressed by increasing the concentration of GDP-mannose in vitro. Thus, either an increased substrate concentration, in vitro, or an increased Vrg4 protein concentration, in vivo, can suppress these vrg4 mutant phenotypes. Vrg4 proteins with alterations in this region were reduced in binding to guanosine 5'-[gamma-(32)P]triphosphate gamma-azidoanilide, a photoaffinity substrate analogue whose binding to Vrg4-HAp was specifically inhibited by GDP-mannose. Taken together, these data are consistent with the model that amino acids in this region of the yeast GDP-mannose transporter mediate the recognition of or binding to nucleotide sugar prior to its transport into the Golgi.     

The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.     

Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.     

Enzyme Cytochemistry of the Alveolar Sacs and Golgian-Like Cisternae in the Ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trosphozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis

Transport of nucleotide sugars across the Golgi apparatus membrane is required for the luminal synthesis of a variety of plant cell surface components. We identified an Arabidopsis gene encoding a nucleotide sugar transporter (designated GONST1) that we have shown by transient gene expression to be localized to the Golgi. GONST1 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), and Golgi-rich vesicles from the complemented strain displayed increased GDP-mannose transport activity. GONST1 promoter::beta-glucuronidase studies suggested that this gene is expressed ubiquitously. The identification of a Golgi-localized nucleotide sugar transporter from plants will allow the study of the importance of this class of proteins in the synthesis of plant cell surface components such as cell wall polysaccharides.     

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

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.     

Reconstitution, identification, and purification of the rat liver golgi membrane GDP-fucose transporter

Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the Golgi apparatus requires the translocation of nucleotide sugars from the cytosol into the lumen of the Golgi. Translocation is mediated by specific nucleotide sugar transporters, integral Golgi membrane proteins that regulate the above glycosylation reactions. A defect in GDP-fucose transport into the lumen of the Golgi apparatus has been recently identified in a patient affected by leukocyte adhesion deficiency type II syndrome (Lubke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) J. Biol. Chem. 274, 25986-25989). We have now identified and purified the rat liver Golgi membrane GDP-fucose transporter, a protein with an apparent molecular mass of 39 kDa, by a combination of column chromatography, native functional size determination on a glycerol gradient, and photoaffinity labeling with 8-azidoguanosine-5'-[alpha-(32)P] triphosphate, an analog of GDP-fucose. The purified transporter appears to exist as a homodimer within the Golgi membrane. When reconstituted into phosphatidylcholine liposomes, it was active in GDP-fucose transport and was specifically photolabeled with 8-azidoguanosine-5'-[alpha-(32)P]triphosphate. Transport was also stimulated 2-3-fold after preloading proteoliposomes with GMP, the putative antiporter.