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.     

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.     

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.     

The preparation of UDP-N-acetylgalactosamine from UDP-N-acetylglucosamine employing UDP-N-acetylglucosamine-4-epimerase

A rapid, simple, and inexpensive method has been developed for preparing UDP-N-acetylgalactosamine in amounts sufficient for several thousand assays of enzymes that employ this nucleotide sugar as substrate. The UDP-N-acetylglucosamine-4-epimerase in extracts of porcine submaxillary glands was used to convert UDP-N-acetylglucosamine to an equilibrium mixture of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (molar ratio, 77:23). The two nucleotide sugars were separated from components in the extract by ion-exchange chromatography and then separated from one another by affinity chromatography on a column of Griffonia simplicifolia lectin I bound to agarose. The UDP-N-acetylgalactosamine was obtained in pure form by ion-exchange chromatography in an overall yield of 91% from the equilibrium mixture. The separation of the two nucleotide sugars by affinity chromatography also provides a rapid assay for the UDPGlcNAc-4-epimerase, which is more accurate and less time consuming than earlier published assays.     

Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei

UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.     

The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 μg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.     

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.     

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.     

The genes for the Golgi apparatus N-acetylglucosaminyltransferase and the UDP-N-acetylglucosamine transporter are contiguous in Kluyveromyces lactis

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked. We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha-D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.     

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.     

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.     

CeHMT-1, a putative phytochelatin transporter, is required for cadmium tolerance in Caenorhabditis elegans

Phytochelatins (PCs), (gamma-Glu-Cys)n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd2+) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, CeHMT-1 satisfies the requirements of a Cd2+ tolerance factor involved in the sequestration and/or elimination of Cd x PC complexes. Heterologous expression of cehmt-1 in S. pombe alleviates the Cd2+-hypersensitivity of hmt- mutants concomitant with the localization of CeHMT-1 to the vacuolar membrane. Suppression of the expression of ce-hmt-1 in intact worms by RNA interference (RNAi) confers a Cd2+-hypersensitive phenotype similar to but more pronounced than that exhibited by ce-pcs-1 RNAi worms. At another level, it is evident from comparisons of the cell morphology of ce-hmt-1 and cepcs-1 single and double RNAi mutants that CeHMT-1 also contributes to Cd2+ tolerance in other ways. Whereas the intestinal epithelial cells of ce-pcs-1 RNAi worms undergo necrosis upon exposure to toxic levels of Cd2+, the corresponding cells of ce-hmt-1 RNAi worms instead elaborate punctate refractive inclusions within the vicinity of the nucleus. Moreover, a deficiency in CeHMT-1 does not interfere with the phenotype associated with CePCS-1 deficiency and vice versa. Double ce-hmt-1; ce-pcs-1 RNAi mutants exhibit both cell morphologies when exposed to Cd2+. These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of CeHMT-1 does not depend exclusively on PC synthesis.     

The conserved oligomeric Golgi complex is required for fucosylation of N-glycans in Caenorhabditis elegans

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.     

Deletion of the intestinal peptide transporter affects insulin and TOR signaling in Caenorhabditis elegans

The mammalian intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides from the gut lumen into intestinal epithelial cells and acts in parallel with amino acid transporters. Here we address the importance of the PEPT1 orthologue PEP-2 for the assimilation of dietary protein and for overall protein nutrition in Caenorhabditis elegans. pep-2 is expressed specifically along the apical membrane of the intestinal cells, and in pep-2 deletion mutant animals, uptake of intact peptides from the gut lumen is abolished. The consequences are a severely retarded development, reduced progeny and body size, and increased stress tolerance. We show here that pep-2 cross-talks with both the C. elegans target of rapamycin (TOR) and the DAF-2/insulin-signaling pathways. The pep-2 mutant enhances the developmental and longevity phenotypes of daf-2, resulting, among other effects, in a pronounced increase in adult life span. Moreover, all aspects of a weak let-363/TOR RNA interference phenotype are intensified by pep-2 deletion, indicating that pep-2 function upstream of TOR-mediated nutrient sensing. Our findings provide evidence for a predominant role of the intestinal peptide transporter for the delivery of bulk quantities of amino acids for growth and development, which consequently affects signaling pathways that regulate metabolism and aging.     

Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.