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Originally published In Press as doi:10.1074/jbc.M504783200 on May 23, 2005

J. Biol. Chem., Vol. 280, Issue 29, 27230-27235, July 22, 2005
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The Human Solute Carrier Gene SLC35B4 Encodes a Bifunctional Nucleotide Sugar Transporter with Specificity for UDP-Xylose and UDP-N-Acetylglucosamine*

Angel Ashikov{ddagger}||, Françoise Routier{ddagger}, Jutta Fuhlrott{ddagger}, Yvonne Helmus§, Martin Wild§, Rita Gerardy-Schahn{ddagger}, and Hans Bakker{ddagger}

From the {ddagger}Zelluläre Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and the §Max Planck Institute for Molecular Biomedicine, Münster and Institute of Cell Biology, Zentrum für Molekularbiologie der Entzündung, University of Münster, Von-Esmarch-Strasse 56, 48148 Münster, Germany

Received for publication, May 2, 2005 , and in revised form, May 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Golgi apparatus is the major compartment involved in the biosynthesis of glycoconjugates (glycoproteins, glycolipids, proteoglycans). Glycosyltransferases that reside in the Golgi apparatus are type II membrane proteins with lumenally oriented catalytic domains (1). Their activity depends on the delivery of nucleotide sugars to the compartmental lumen, a function accomplished by nucleotide sugar transporters (NSTs)1 (for a review, see Ref. 2).

The distribution patterns of NSTs and transporters of other substrates needed in posttranslational modifications in the Golgi (e.g. PAPS and ATP) have been intensively investigated using purified vesicles in the presence of radioactively labeled substrates (3-8). In these studies, Golgi vesicles isolated from mammalian cells were shown to transport UDP-Glc, UDP-Gal, UDP-Xyl, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, CMP-Sia, GDP-Fuc, ATP, and PAPS (for a review, see Ref. 9). Further biochemical characterization of the transport proteins demonstrated that they act as antiporters exchanging the substrates for the respective mono-nucleotides (7, 10).

A breakthrough came with the cloning of the first NSTs. By complementation cloning in characterized cell mutants, the UDP-GlcNAc transporter was cloned from yeast (11), the CMP-Sia and UDP-Gal transporters were cloned from mammals (12, 13), and the GDP-Man transporter was cloned from the protozoan Leishmania (14). All isolated NST genes encoded homologous type III membrane proteins. Ma et al. (14) noted first that the gene family not only included NSTs but also proteins of the inner chloroplast membrane transporting other metabolites (15, 16). Nowadays, this gene family is known as the drug-metabolite transporter superfamily, which comprises solute transporters from both prokaryotes and eukaryotes (17).

NSTs preserve their specificity when expressed in heterologous systems. Initially demonstrated for the murine CMP-Sia transporter by Berninsone et al. (18), this aspect has been used later to clone NSTs from different species. For instance, the canine UDP-GlcNAc transporter was cloned by complementation of a transport-deficient Klyveromyces lactis strain (11), and the Caenorhabditis elegans GDP-Fuc transporter (19) was cloned by complementation of human fibroblasts isolated from a patient with congenital disorder of glycosylation type IIc. More recently, two UDP-Gal transporters from Arabidopsis (20) have been identified by complementation of the Chinese Hamster Ovary (CHO) cell mutant Lec8 (21). Together, the architectural conservation, which allows the identification of putative NSTs in the gene data bases, and the functional conservation, which allows specificity testing in a low background system such as Saccharomyces cerevisiae (22), have accelerated the cloning and functional characterization of NSTs. Unexpectedly, the testing of heterologously expressed NSTs demonstrated that many of these proteins transport more than one substrate. Functional activities determined for human NSTs are listed in Table II.


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  		TABLE II
Members of the solute carrier family 35 (SLC35) are summarized and their specificity is indicated

Sequences investigated in the current study are shaded. The phylogenetic analysis demonstrates splitting into six subfamilies.

 
In addition, the table highlights all human members of the drug-metabolite transporter family (17), which have been recently assembled in the solute carrier family 35 (SLC35) (23). Besides characterized NSTs, this family comprises the PAPS transporter and numerous genes with unknown functions.

Here we describe the characterization of gene SLC35B4 as encoding a Golgi localized NST with dual specificity for UDP-Xyl and UDP-GlcNAc. With this first molecular characterization of a UDP-Xyl transport protein, we confirmed earlier biochemical data (24, 25) that UDP-Xyl can access the Golgi apparatus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Radioactive nucleotide-sugars UDP-[1-3H]Glc, UDP-[1-3H]Gal, UDP-[6-3H]GlcNAc, UDP-[1-3H]GalNAc, GDP-[2-3H]Fuc, CMP-[9-3H]NeuAc, UDP-[14C(U)]GlcA, and UDP-[14C(U)]Xyl were purchased from PerkinElmer Life Sciences. Cold Nucleotide sugars were from Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan, except for UDP-GalNAc, UDP-GlcA, and UDP-Xyl, which were from Sigma. Zymolyase 100T was obtained from ICN Biomedicals (Aurora, OH), and monoclonal anti-mouse antibody M5 directed against the FLAG sequence MDYKDDDDK was from Sigma. A rabbit antiserum against the catalytic domain of the {alpha}-mannosidase II (26) was a kind gift of Dr. Kelly Moremen, University of Georgia, Athens, GA.

Multiple Alignments, Transmembrane Domain Prediction, and Phylogenetic Analysis—Human members of the SLC35 family were identified by WU-BLAST2 searches in the protein databases using various known members of the drug-metabolite transporter superfamily as input. Searches were done at protein level and limited to the proteins predicted from the human genomic sequence. The TMHMM server v 2.0 was used for prediction of transmembrane helices in protein (Center for Biological Sequence Analysis, Technical University of Denmark). The multiple alignment and building of the phylogenetic tree of members of the human SLC35 family was done using the online clustalW program at the EMBL-EBI (Hinxton, UK).

Yeast Strains and Plasmids—S. cerevisiae strain YPH500 (MAT{alpha} ura3-52 lys2-801 ade2-101 trp1-{Delta} 63 his3-{Delta}200 leu2-{Delta}1) and BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0; YEL004w::kanMX4 (EUROSCARF, European S. cerevisiae Archive for Functional Analysis; Frankfurt, Germany) were used for yeast expression experiments. A copper-inducible yeast expression vector was constructed by replacing the Gal-inducible promoter in pYES2/NT-C (Invitrogen) by the CUP1 promoter of pYEX-BESN (27). pYEX-BESN was a generous gift from Dr. Masao Kawakita (Tokyo Metropolitan Institute of Medical Science, Japan). The primers 5'-GCT TAC TAG TCT TTT GCT GGC ATT TCT TTT AGA AGC AAA AAG-3' and 5'-TAC TAA GCT TCC AAT TCG CTG AAT ATT TTA TG-3', containing SpeI and HindIII restriction sites, respectively, were used to amplify the CUP1 promoter from pYEX-BESN (28). The resulting PCR fragment was digested and subcloned into pYES2/NT-C. The His tag encoding sequence contained in pYES2 was replaced by either the linker 5'-AGC TTA CCA TGG ACT ACA AGG ACG ATG ACG ATA AGG TAC-3' after HindIII/KpnI digestion or the linker 5'-AGC TTA CCA TGG ACT ACA AGG ACG ATG ACG ATA AGG-3' after HindIII/EcoRI digestion, resulting in the vectors pYEScupFLAGK and pYEScupFLAGE, respectively. These vectors drive the expression of N-terminally FLAG-tagged proteins.

Human members of the SLC35 family were amplified by PCR from I.M.A.G.E. clones (I.M.A.G.E. Consortium (29) obtained via RZPD, Berlin, Germany) and cloned in pYEScupFLAGK using KpnI or BamHI at the 5' side and XbaI at the 3' side or in pYEScupFLAGE using EcoRI at the 5' side and XbaI at the 3' side. Table I lists the identification numbers of I.M.A.G.E. clones, the systematic gene names as allocated in the SLC35 family, primers and restriction sites used to amplify and clone the putative NST cDNAs, and names of resulting vector constructs.


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  		TABLE I
Vector constructs containing SLC35 family members used in this study

 
Yeast Transformation, Golgi Preparation, and in Vitro Transport Assay—Yeast cells were transformed using the lithium acetate method provided by Invitrogen. After transformation, yeast cells were cultured on selective medium containing 0.67% Bacto-yeast nitrogen base without amino acids but supplemented with L-leucine, L-histidine, L-tryptophan, L-lysine, adenine, and 2% glucose. Cells were grown to A600 0.8, supplemented with copper-sulfate to a final concentration of 0.5 mM, and cultured for another 2 h.

The subcellular fractionation of yeast cells and the in vitro transport assays were performed essentially as described (28, 30). Therefore, cultured cells were harvested by centrifugation (5 min at 1,500 x g) and washed twice with ice-cold 10 mM NaN3. The weight of wet cells was measured, and cells were resuspended in zymolyase buffer (3 ml/g of cells; 50 mM potassium phosphate, pH 7.5; 1.4 M sorbitol; 10 mM NaN3 and 0.3% {beta}-mercaptoethanol) containing 0.6 mg/ml of zymolyase-100T. The suspension was incubated for 20 min at 30 °C. Spheroplasts were collected by centrifugation (5 min at 1,000 x g) and resuspended in lysis buffer (4 ml/g of cells; 10 mM Hepes-Tris, pH 7.4; 0.8 M sorbitol; 1 mM EDTA) containing complete EDTA-free protease inhibitor mixture (Roche Applied Science). After homogenization with 10 strokes in a Dounce homogenizer, the lysate was centrifuged (5 min, 1,500 x g) to remove unlysed cells and debris. Endoplasmic reticulum- and Golgi-rich fractions were then obtained by centrifugation at 10,000 x g for 10 min (endoplasmic reticulum) and 100,000 x g for 1 h (Golgi). The 100,000 x g pellet was carefully resuspended in lysis buffer (0.8 ml/g of cells), and aliquots of 100 µl were snap-frozen and kept at -80 °C. Protein concentrations were determined using the BCATM kit (Pierce).

For transport assay reactions, equal volumes (50 µl of each) of 2 mM radioactive nucleotide sugar (2,000-4,000 dpm/pmol) in assay buffer (10 mM Tris-HCl, pH 7.0; 0.8 M sorbitol; 2 mM MgCl2) and vesicle preparation (equivalent to 75-100 µg of protein) were incubated for 30 s at 30 °C. Reactions were stopped by dilution with 1 ml of assay buffer containing 1 µM respective cold nucleotide sugar. The separation of vesicles and nucleotide sugars was achieved by filtration trough nitrocellulose filter (MFTM membrane filters Millipore, Bedford, MA). Filters were washed three times with 2 ml of ice-cold assay buffer containing the corresponding cold nucleotide sugar at a concentration of 1 µM, and radioactivity associated with the vesicular fraction was measured by liquid scintillation in a LS 5000CE counter (Beckman Coulter). Golgi vesicles from yeast cells transformed with an empty vector were used to measure endogenous transport. The endogenous transport of UDP-Glc was used to control the quality of vesicles. Transport values for each construct were calculated as mean from two independent experiments using independently prepared membrane fractions. In each experiment, nucleotide sugars were assayed in duplicate with the same vesicle preparation.

Mammalian Cell Lines, Transfection, and Staining Procedures—Fibroblasts defective in the Golgi GDP-Fuc transport isolated from a leukocyte adhesion deficiency type II patient (19) were cultured and transfected as described (31) using the human dermal fibroblast nucleo-factor kit (Amaxa Biosystems, Cologne, Germany). 48 h after transfection, complementation of cells was monitored by staining with the fucose-specific Aleuria aurantia lectin (Vector Laboratories, Burlingame, CA).

The CHO cell lines Lec8 (21, 32) and 6B2, a subclone of the Lec2 complementation group (12), were transfected using Lipofectamine (Invitrogen) following the manufacturer's instructions. Complementation of Lec8 and 6B2 cells was determined by cell surface immunostaining as described in Refs. 12 and 20.

Studies to determine the subcellular localization of the recombinant FLAG-SLC35B4 in mammalian cells were carried out with CHO-K1 cells (ATCC CCL-61). Cells were seeded onto glass coverslips, transfected with Lipofectamine as described above, and analyzed by immunofluorescence. Therefore, cells 2 days after transfection were fixed in 4% paraformaldehyde and permeabilized for 30 min with 0.1% saponin in phosphate-buffered saline containing 0.1% bovine serum albumin. After the permeabilization step, samples were incubated with the respective primary antibodies (anti-FLAG tag M5 and rabbit anti-{alpha}-mannosidase II). After three washings (phosphate-buffered saline, 0.1% bovine serum albumin, 0.1% Tween 20), cells were incubated with anti-mouse IgCy3 and anti-rabbit Ig-Alexa 488 for 1 h at room temperature. After three additional washes in phosphate-buffered saline, 0.1% bovine serum albumin, 0.1% Tween 20 and a final wash in phosphate-buffered saline, slides were briefly rinsed in water, mounted in Mowiol, and analyzed under a Leica DM IRBE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Putative Human Nucleotide Sugar Transporters—When this study was started, six out of the expected eight Golgi nucleotide sugar transport activities had been identified (12, 13, 19, 28, 33-35), and it had been highlighted that NST form a family of structurally conserved proteins. Characterized members of the drug-metabolite transporter family were, therefore, used as templates to search the human genome data bases for the existence of additional homologous sequences. The cut-off was set for proteins with a length sufficient to accommodate the 10 transmembrane domains expected for NSTs (36). In these searches, we identified 17 sequences of which five were known. Sequences identified in our search are displayed in Table II as part of the recently described SLC35 family (23). All, except clone C2ORF18, overlay with listed SLC35 family members. A phylogenetic analysis carried out including the newly identified gene C2ORF18 demonstrates that it is a sixth member of subfamily SLC35F (Table II), which is most homologous to the plant purine transporters (37). While this study was in progress, the genes with the numbers SLC35B2 and SLC35D2 have been shown to represent the PAPS transporter (38, 39) and a multispecific Golgi localized NST (40, 41), respectively.

Functional Characterization of SLC35 Family Members—To identify NSTs with transport activities for UDP-Glc and UDP-Xyl, we decided to heterologously express the candidate genes in S. cerevisiae and measure transport of the nucleotide sugars in vitro with Golgi fractions isolated from transformed cells. PCR fragments harboring the complete open reading frames were subcloned into modified pYES2/NT-C expression vectors downstream of a FLAG sequence tag to enable detection of recombinant proteins (see "Experimental Procedures" and Table I). The characterized gene SLC35A2 (UDP-Gal/UDP-Gal-NAc transporter (13, 28), cloned into the same vector system, was used as a control. For 9 out of the 12 newly identified gene family members, we were able to clone the complete open reading frames from EST clones. Obtained constructs are listed and described in Table I, and gene names are shaded in Table II.

Proteins were expressed in the S. cerevisiae strain YPH500, and Golgi-rich microsomal fractions were isolated and used to measure transport activity with radioactively labeled sugar nucleotides. The Western blot shown in Fig. 1C demonstrates that all proteins were expressed. None of the expressed proteins was, however, able to increase UDP-Glc transport significantly over the high endogenous transport level (Fig. 1A). In contrast, endogenous transport of UDP-Xyl was virtually absent in vesicles isolated from mock-transfected YPH500 cells (Fig. 1B; background 1.1 pmol/mg/min) but rose to 6.8 pmol/mg/min in the vesicle fraction isolated from SLC35B4 transformed cells. All other candidate genes, including the characterized human UDP-Gal/UDP-GalNAc transporter, did not significantly change UDP-Xyl background transport.



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  		FIG. 1.
UDP-Glc and UDP-Xyl transport activity of members of the SLC35 family. A and B, UDP-Glc (A) and UDP-Xyl (B) transport activity of Golgi vesicles isolated from yeast cells transformed with either the empty vector control (C) or vector constructs containing FLAG-tagged members of the human SLC35 gene family. Each value represents the average of two test series carried out with independent Golgi preparations. C, Western blot analysis was used to control the expression of recombinant proteins. Golgi vesicles of transformed cells were lysed, separated in SDS-PAGE, and after transfer to nitrocellulose, stained with the anti-FLAG tag antibody M5.

 
SLC35B4 Encodes an NST with Transport Activity for UDP-Xyl and UDP-GlcNAc—Because many of the NSTs cloned and functionally characterized today exhibit transport activity for more than a single nucleotide sugar, we decided to analyze SLC35B4 for additional substrates. As shown in Fig. 2, a transport assay was carried out with seven radioactively labeled nucleotide sugars. The high endogenous transport of UDP-Glc demonstrated the high quality of the vesicle preparation. Although no transport activity was found with the UDP-activated sugars Gal, GalNAc, and GlcA, the presence of FLAG-SLC35B4 clearly increases the transport activity for UDP-Xyl (6.2-fold vector control) and UDP-GlcNAc (2.6-fold vector control). In the case of GDP-Fuc, rather high transport values were observed already in vesicles isolated from mock-transfected cells. Transport values were, however, not altered in the presence of FLAG-SLC35B4, thus arguing against the recognition and transport of GDP-Fuc. The presented data identify the human SLC35B4 gene as dually active NST with specificity for UDP-Xyl and UDP-GlcNAc.

Because complementation cloning in characterized mutant cell lines provides a standard technique in our laboratory, potential transport capabilities of SLC35B4 were additionally tested in existing mutants. Included in this test were (i) CHO 6B2 cells, which cells belong to the Lec2 complementation group defective in the CMP-Sia transporter (12); (ii) CHO Lec8 cells, which are defective in the UDP-Gal transporter (21, 32); and (iii) human CDGIIc fibroblasts, which are defective in the GDP-Fuc transporter (42). The vector construct pcDNA3FLAGKB4 (Table I) driving the expression of an N-terminally FLAG-tagged protein was used in transient transfection studies. Although complementation of the respective cells was achieved with the positive controls (SLC35A1, SLC35A2, and SLC35C1 cloned into the same vector), and indirect immunofluorescence with the anti-FLAG tag antibody M5 demonstrated expression of FLAG-SLC35B4 in all cell lines, the latter was unable to restore the wild type glycosylation patterns. These data exclude the transport of CMP-Sia, UDP-Gal, and GDP-Fuc by SLC35B4.

Confirmation of UDP-Xyl and UDP-GlcNAc Transport Activity—Although the data presented so far typify SLC35B4 as a dual transporter for UDP-Xyl and UDP-GlcNAc, the endogenous transport for UDP-GlcNAc did not allow us to directly compare the two transport activities. Since the gene encoding the UDP-GlcNAc transporter in S. cerevisiae is known (43) and not essential for yeast growth, this presumed zero background system was used to compare the two transport capabilities of SLC35B4. Results are shown in Fig. 3. As expected, the endogenous UDP-GlcNAc transport was reduced to background level in the mock-transfected BY4741 knock-out strain but increased by a factor of 7.5 in Golgi vesicles isolated from cells transfected with FLAG-SLC35B4. The factor of 6.5 measured for the increase in UDP-Xyl transport is identical to that already determined in the wild type strain YPH500 (compare Fig. 2). These data demonstrating comparable activity for UDP-Xyl and UDP-GlcNAc proved the dual activity of the newly identified NST.

FLAG-tagged SLC35B4 Is a Golgi-resident Protein—Finally, the subcellular localization of the expressed epitope-tagged protein was determined by indirect immunofluorescence in CHO cells. To this end, cells were transfected with pcDNA3FLAGKB4, and parallel samples were stained with the anti-FLAG-antibody M5 and the {alpha}-mannosidase II antibody as a Golgi marker (26, 36). The identical perinuclear staining (Fig. 4) obtained with both control and probe antibody clearly demonstrates Golgi localization of this nucleotide sugar transporter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we describe the identification of a bifunctional NST exhibiting transport activity for UDP-Xyl and UDP-GlcNAc. Although NSTs transporting UDP-GlcNAc as a single activity (11, 33, 44) or as one of several activities (40, 41, 45) have been characterized from different organisms, this is the first identification of a UDP-Xyl transport protein. In humans, the gene with the systematic name SLC35B4 is mapped to chromosome 7q33 and contains 10 exons and large 5'- and 3'-untranslated regions. The predicted protein is 331 amino acids long and has a calculated molecular mass of 37,422.6 Da. Topological analyses predict the presence of 10 hydrophobic transmembrane regions, which is in accordance with the number experimentally determined for the CMP-sialic acid transporter (36).

Immunofluorescence studies carried out with the FLAG-tagged protein localized the protein exclusively to the Golgi apparatus. Our finding confirmed previous biochemical studies, which demonstrated (i) UDP-Xyl uptake into isolated Golgi vesicles (24, 25) or (ii) [14C]Xyl integration into glycoconjugates in chondrocytes permeabilized and treated with UDP-[14C]Xyl (46). Still, the physiological meaning behind UDP-Xyl transport into the Golgi apparatus of mammalian cells is not clear, particularly because UDP-Xyl can be generated by decarboxylation of UDP-GlcA inside the compartmental lumen (46). The responsible activity, the UDP-xylose synthase, was known for long to associate with microsomal membranes (47). Cloning of the human UDP-xylose synthase succeeded recently. The cloned sequence encodes a type II transmembrane protein with a predicted luminal catalytic domain (48).



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  		FIG. 2.
Transport activity of SLC35B4 with different nucleotide sugars. Gray bars represent control values carried out with Golgi fractions isolated from vector transformed yeast cells. Black bars represent the activity of Golgi preparations isolated from yeast cells expressing FLAG-tagged SLC35B4. Each value represents the average of two independent experiments. In each experimental series, values were measured in duplicates.

 



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  		FIG. 3.
UDP-GlcNAc and UDP-Xyl transport activity measured after expression of the FLAG-tagged SLC35B4 in a yeast strain defective in UDP-GlcNAc transport. Assays were performed as described in the legend for Fig. 2.

 



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  		FIG. 4.
Intracellular localization of the SLC35B4 protein. CHO cells were transiently transfected with FLAG-tagged SLC35B4, and the localization of the recombinant protein was analyzed by indirect immunofluorescence microscopy. The double functional UDP-Xyl/GlcNAc transporter (red) co-localizes with the Golgi marker mannosidase II (green).

 
Besides its importance as an initiating sugar in proteoglycan biosynthesis, xylose is also found in a trisaccharide (Xyl-Xyl-Glc) that decorates functionally important proteins. This epitope has been identified in Notch (49), a key factor in the regulation of cell growth and differentiation during ontogeny (50) and in the blood coagulation factors VII and IX (51). Although direct evidence is missing, SLC35B4 could be involved in the biosynthesis of these O-linked glycans. In fact, the expression of the gene is relatively high in liver (52), in which the coagulation factors VII and IX are produced. Nevertheless, all these considerations depend on the existence of UDP-Xyl pools in the cytoplasm of mammalian cells that would justify the need of an UDP-Xyl transport protein. In plants, which have UDP-xylose synthase activity in the cytoplasm and in endoplasmic reticulum and Golgi lumen (53, 54), this pool seems to exist. No information is, however, available for the mammalian cell. An alternative for de novo synthesis of nucleotide sugars is the salvage pathway in which nucleotide sugars can be generated from sugars taken up from outside or formed during degradation of glycans. Again, in plants, it has been shown that UDP-Xyl can be generated in the cytoplasm from xylose-1-phosphate (55). Future studies will show whether this pathway also exists in mammals.

Initially unexpected but obvious after the functional characterization of numerous NSTs is the fact that many of these proteins have more than a single specificity (Table II) (22, 28, 41, 56-60). Out of the eight characterized NSTs in the SLC35 family, three are able to transport UDP-GlcNAc (Table II). Because UDP-GlcNAc transport activity has been found to be combined with substrates as different as UDP-Gal (59) and UDP-Xyl (this study), the prediction of sugar-specific epitopes that enable the transport becomes very difficult. Moreover, linear alignment of all known UDP-GlcNAc transporters did not reveal a common motif, which may point toward UDP-GlcNAc specificity. In line with this, the phylogenetic tree in Table II demonstrates that UDP-GlcNAc transporting proteins belong to very distant subfamilies (e.g. SLC35A, SLC35B, and SLC35D). A possible explanation still is that the nucleotide part is most important in defining substrate specificity. In accordance with this, no cross-transport of differentially activated sugars has been identified so far. Kawakita and co-workers (30, 61) were, however, able to generate bifunctional chimeras by exchanging domains between the closely related human CMP-Sia (SLC35A1) and UDP-Gal (SLC35A2) transporters.

Recently, purification and functional reconstitution in artificial liposomes has succeeded for the recombinant Leishmania LPG2 protein (60). This study provided not only a major step toward obtaining structural information of NSTs but also the ultimate system to determine the genuine transport activity of these proteins. Using the zero background conditions, the study confirmed the specificity of LPG2 (14) for three substrates, GDP-Man, GDP-Fuc, and GDP-Ara. Similarly, our study demonstrated that direct comparison of transport activities requires a zero background system, which in the current study was obtained by a yeast strain with inactivated endogenous UDP-GlcNAc transport (compare Figs. 2 and 3).

Not finally resolved is the question of how UDP-Glc is transported into the Golgi apparatus. UDP-Glc is synthesized in the cytoplasm (62) and requires a transport system. The multifunctional transporter SLC35D2 has been suggested to recognize and transport UDP-Glc (41). SLC35B4 is a second promising candidate because a transporter able to recognize UDP-GlcNAc and UDP-Xyl should also transport UDP-Glc (63). The high endogenous transport of UDP-Glc in yeast, however, limits the conclusive determination of specific UDP-Glc transport of recombinantly expressed transporters. Therefore, it is an important goal in our laboratory to generate a model system with reduced background activity.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ971941 [GenBank]

* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to R. G.-S. and M. W.) and the Max Planck Society (to M. W. and Y. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| A Ph.D student in the GK-745 financed by the Deutsche Forschungsgemeinschaft. Back

To whom correspondence should be addressed. Tel.: 49-511-532-9802; Fax: 49-511-532-8801; E-mail: Gerardy-schahn.rita{at}mh-hannover.de.

1 The abbreviations used are: NST, nucleotide sugar transporter; CHO, Chinese hamster ovary; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; SLC35, solute carrier family 35. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Nobuhiro Ishida and Masao Kawakita, Tokyo, Japan, for the gift of pYEX-BESN and helpful advice for transport assays and Dr. Lothar Elling, Aachen, Germany, for UDP-xylose.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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