Publications

• Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3.

  Toscanini M.A., Favarolo M.B., Gonzalez Flecha F.L., Ebert B., Rautengarten C., Bredeston L.M.

  Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nu ... >> More

  Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an E<i>XX</i>K motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein. << Less

  J. Biol. Chem. 294:10042-10054(2019) [PubMed] [EuropePMC]

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

  Ishida N., Yoshioka S., Chiba Y., Takeuchi M., Kawakita M.

  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 ... >> More

  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. << Less

  J. Biochem. 126:68-77(1999) [PubMed] [EuropePMC]

• Variety of nucleotide sugar transporters with respect to the interaction with nucleoside mono- and diphosphates.

  Muraoka M., Miki T., Ishida N., Hara T., Kawakita M.

  Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochem ... >> More

  Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed. << Less

  J. Biol. Chem. 282:24615-24622(2007) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• 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.

  Aoki K., Ishida N., Kawakita M.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 278:22887-22893(2003) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.

• A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia.

  Rautengarten C., Quarrell O.W., Stals K., Caswell R.C., De Franco E., Baple E., Burgess N., Jokhi R., Heazlewood J.L., Offiah A.C., Ebert B., Ellard S.

  We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene ... >> More

  We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum. << Less

  Hum. Mol. Genet. 28:3543-3551(2019) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP.

  Waldman B.C., Rudnick G.

  We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the condition ... >> More

  We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the conditions used, extravesicular degradation of UDP-GlcNAc has been eliminated, and less than 15% of the internalized radioactivity becomes associated with endogenous macromolecules. Of the remaining intravesicular label, 85% remains unmetabolized UDP-[3H]GlcNAc, and approximately 15% is hydrolyzed to [3H]GlcNAc-1-phosphate. Efflux of accumulated UDP-[3H]GlcNAc is induced by addition of UMP, UDP, or UDP-galactose to the external medium. Permeabilization of Golgi vesicles causes a rapid and nearly complete loss of internal UDP-[3H]GlcNAc, indicating that the results reflect transport and not binding. Moreover, transport of UDP-[3H]GlcNAc into these Golgi vesicles was stimulated up to 5-fold by mechanically preloading vesicles with either UDP-GlcNAc or UMP. The response of UMP/UMP exchange and UMP/UDP-GlcNAc exchange to alterations in intravesicular and extravesicular pH suggests that UDP-GlcNAc enters the Golgi apparatus in electroneutral exchange with the dianionic form of UMP. << Less

  Biochemistry 29:44-52(1990) [PubMed] [EuropePMC]