Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.     

Substrate recognition by nucleotide sugar transporters: further characterization of substrate recognition regions by analyses of UDP-galactose/CMP-sialic acid transporter chimeras and biochemical analysis of the substrate specificity of parental and chimeric transporters

Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.     

Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose.

A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT). When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells. DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms. Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal. When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal. One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product. However, this mutation was suppressed by an Ile51 to Thr second-site mutation. Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule.     

Short N-terminal region of UDP-galactose transporter (SLC35A2) is crucial for galactosylation of N-glycans

UDP-galactose transporter (UGT) and UDP-N-acetylglucosamine transporter (NGT) form heterologous complexes in the Golgi apparatus (GA) membrane. We aimed to identify UGT region responsible for galactosylation of N-glycans. Chimeric proteins composed of human UGT and either NGT or CMP-sialic acid transporter (CST) localized to the GA, and all but UGT/CST chimera corrected galactosylation defect in UGT-deficient cell lines, although at different efficiency. Importantly, short N-terminal region composed of 35 N-terminal amino-acid residues of UGT was crucial for galactosylation of N-glycans. The remaining molecule must be derived from NGT not CST, confirming that the role played by UGT and NGT is coupled.     

Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.     

Endoplasmic reticulum retention of the large splice variant of the UDP-galactose transporter is caused by a dilysine motif

Nucleotide-sugar transporters supply mainly the Golgi glycosyltransferases with substrates. Some glycosyltransferases in the endoplasmic reticulum (ER), however, also use activated sugars. Recent studies have demonstrated that UDP-galactose (UDP-Gal) is the substrate for the ER resident ceramide-galactosyltransferase (cer-GalT) and cells expressing cer-GalT are able to retain the UDP-Gal transporter (UGT) by physical contacts formed between the two proteins. Here, we describe a second active mechanism for ER localization of the UGT. The UGT is produced in two splice forms UGT1 and UGT2. The proteins vary only at their extreme C-termini but show strikingly different intracellular distribution. Although N-terminally epitope tagged forms of UGT1 localize exclusively to the Golgi, similar constructs of UGT2 show both ER and Golgi localization. The dilysine motif KVKGS contained in UGT2 can be demonstrated to be responsible for the dual localization because: (1) disturbance of the signal via site specific mutation or C-terminal extension completely shifts the transporter to the Golgi, (2) transfer of the dilysine motif is sufficient to redistribute the Golgi CMP-sialic acid transporter to the ER, and (3) replacement of KVKGS by the strong ER retention signal KKNT is sufficient to completely retain UGT2 in the ER.     

A CMP-sialic acid transporter cloned from Arabidopsis thaliana

Sialylation of glycans is ubiquitous in vertebrates, but was believed to be absent in plants, arthropods, and fungi. However, recently evidence has been provided for the presence of sialic acid in these evolutionary clades. In addition, homologs of mammalian genes involved in sialylation can be found in the genomes of these taxa and for some Drosophila enzymes, involvement in sialic acid metabolism has been documented. In plant genomes, homologs of sialyltransferase genes have been identified, but there activity could not be confirmed. Several mammalian cell lines exist with defects in the sialylation pathway. One of these is the Chinese hamster ovary cell line Lec2, deficient in CMP-sialic acid transport to the Golgi lumen. These mutants provide the possibility to clone genes by functional complementation. Using expression cloning, we have identified an Arabidopsis thaliana nucleotide sugar transporter that is able to complement the CMP-sialic acid transport deficiency of Lec2 cells. The isolated gene (At5g41760) is a member of the triose-phosphate/nucleotide sugar transporter gene family. Recombinant expression of the gene in yeast and testing in vitro confirmed its ability to transport CMP-sialic acid.     

Membrane topology of the mammalian CMP-sialic acid transporter

Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.     

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.     

A single Caenorhabditis elegans Golgi apparatus-type transporter of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine

The genome of Caenorhabditis elegans encodes for 18 putative nucleotide sugar transporters even though its glycome only contains 7 different monosaccharides. To understand the biological significance of this phenomenon, we have begun a systematic substrate characterization of the above putative transporters and have determined that the gene ZK896.9 encodes a Golgi apparatus transporter for UDP-glucose, UDP-galactose, UDP- N-acetylglucosamine, and UDP- N-acetylgalactosamine. This is the first tetrasubstrate nucleotide sugar transporter characterized for any organism and is also the first nonplant transporter for UDP-glucose. Evidence for the above substrate specificity and substrate transport saturation kinetics was obtained by expression of ZK896.9 in Saccharomyces cerevisiae followed by Golgi enriched vesicle isolation and assays in vitro. Further evidence for UDP-glucose transport was obtained by expression of ZK 896.9 in Giardia lamblia, an organism recently characterized as having endogenous transport activity for only UDP- N-acetylglucosamine. Expression of ZK896.9 was also able to correct the phenotype of a mutant Chinese ovary cell line specifically defective in the transport of UDP-galactose into the Golgi apparatus and of a mutant of the yeast Kluyveromyces lactis specifically defective in the transport of UDP- N-acetylglucosamine into its Golgi apparatus. Because up to now all three other characterized nucleotide sugar transporters of C. elegans have been found to transport two or three substrates, the substrate specificity of ZK896.9 raises questions as to the evolutionary ancestry of this group of proteins in this nematode.     

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCA^r and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCA^r mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCA^r mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway.     

Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.     

Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

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-side-out 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 Golgi-enriched 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 UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

The Golgi CMP-sialic acid transporter: A new CHO mutant provides functional insights

A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.     

The CMP-sialic acid transporter is localized in the medial-trans Golgi and possesses two specific endoplasmic reticulum export motifs in its carboxyl-terminal cytoplasmic tail

The addition of sialic acid to glycoproteins and glycolipids requires Golgi sialyltransferases to have access to their glycoconjugate substrates and nucleotide sugar donor, CMP-sialic acid. CMP-sialic acid is transported into the lumen of the Golgi complex through the CMP-sialic acid transporter, an antiporter that also functions to transport CMP into the cytosol. We localized the transporter using immunofluorescence and deconvolution microscopy to test the prediction that it is broadly distributed across the Golgi stack to serve the many sialyltransferases involved in glycoconjugate sialylation. The transporter co-localized with ST6GalI in the medial and trans Golgi, showed partial overlap with a medial Golgi marker and little overlap with early Golgi or trans Golgi network markers. Endoplasmic reticulum-retained forms of sialyltransferases did not redistribute the transporter from the Golgi to the endoplasmic reticulum, suggesting that transporter-sialyltransferase complexes are not involved in transporter localization. Next we evaluated the role of the transporter's N- and C-terminal cytoplasmic tails in its trafficking and localization. The N-tail was not required for either endoplasmic reticulum export or Golgi localization. The C-tail was required for endoplasmic reticulum export and contained di-Ile and terminal Val motifs at its very C terminus that function as independent endoplasmic reticulum export signals. Deletion of the last four amino acids of the C-tail (IIGV) eliminated these export signals and prevented endoplasmic reticulum export of the transporter. This form of the transporter supplied limited amounts of CMP-sialic acid to Golgi sialyltransferases but was unable to completely rescue the transporter defect of Lec2 Chinese hamster ovary cells.     

Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter.

We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-K1 cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GlcNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GlcNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GlcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.