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. 1988 Sep;82(3):1113–1122. doi: 10.1172/JCI113668

Carrier-mediated translocation of uridine diphosphate glucose into the lumen of endoplasmic reticulum-derived vesicles from rat liver.

F Vanstapel 1, N Blanckaert 1
PMCID: PMC303626  PMID: 3417868

Abstract

Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bansal S. K., Gessner T. A unified method for the assay of uridine diphosphoglucuronyltransferase activities toward various aglycones using uridine diphospho[U-14C]glucuronic acid. Anal Biochem. 1980 Dec;109(2):321–329. doi: 10.1016/0003-2697(80)90655-7. [DOI] [PubMed] [Google Scholar]
  2. Barthelson R., Roth S. Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose. Biochem J. 1985 Jan 1;225(1):67–75. doi: 10.1042/bj2250067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretz R., Stäubli W. Detergent influence on rat-liver galactosyltransferase activities towards different acceptors. Eur J Biochem. 1977 Jul 1;77(1):181–192. doi: 10.1111/j.1432-1033.1977.tb11656.x. [DOI] [PubMed] [Google Scholar]
  4. Capasso J. M., Hirschberg C. B. Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7051–7055. doi: 10.1073/pnas.81.22.7051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carey D. J., Sommers L. W., Hirschberg C. B. CMP-N-acetylneuraminic acid: isolation from and penetration into mouse liver microsomes. Cell. 1980 Mar;19(3):597–605. doi: 10.1016/s0092-8674(80)80036-5. [DOI] [PubMed] [Google Scholar]
  6. Eriksson L. C., Bergstrand A., Dallner G. Lateral enzyme topology in the rough endoplasmic reticulum of rat liver. Cell Tissue Res. 1977 Mar 30;179(1):17–43. doi: 10.1007/BF00278461. [DOI] [PubMed] [Google Scholar]
  7. Faltynek C. R., Silbert J. E., Hof L. Inhibition of the action of pyrophosphatase and phosphatase on sugar nucleotides. J Biol Chem. 1981 Jul 25;256(14):7139–7141. [PubMed] [Google Scholar]
  8. Finch S. A., Slater T. F., Stier A. Nucleotide metabolism by microsomal UDP-glucuronyltransferase and nucleoside diphosphatase as determine by 31P nuclear-magnetic-resonance spectroscopy. Biochem J. 1979 Mar 1;177(3):925–930. doi: 10.1042/bj1770925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fleischer B. Mechanism of glycosylation in the Golgi apparatus. J Histochem Cytochem. 1983 Aug;31(8):1033–1040. doi: 10.1177/31.8.6345657. [DOI] [PubMed] [Google Scholar]
  10. Fleischer B. The nucleotide content of rat liver Golgi vesicles. Arch Biochem Biophys. 1981 Dec;212(2):602–610. doi: 10.1016/0003-9861(81)90403-3. [DOI] [PubMed] [Google Scholar]
  11. Fleischer S., Kervina M. Subcellular fractionation of rat liver. Methods Enzymol. 1974;31:6–41. doi: 10.1016/0076-6879(74)31005-1. [DOI] [PubMed] [Google Scholar]
  12. Godelaine D., Beaufay H., Wibo M. Incorporation of N-acetylglucosamine into endogenous acceptors of rough microsomes from rat liver: stimulation by GTP after treatment with pyrophosphate. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1095–1099. doi: 10.1073/pnas.74.3.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hochman Y., Kelley M., Zakim D. Modulation of the number of ligand binding sites of UDP-glucuronyltransferase by the gel to liquid-crystal phase transition of phosphatidylcholines. J Biol Chem. 1983 May 25;258(10):6509–6516. [PubMed] [Google Scholar]
  14. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  15. Kuhn N. J., White A. Evidence for specific transport of uridine diphosphate galactose across the Golgi membrane of rat mammary gland. Biochem J. 1976 Jan 15;154(1):243–244. doi: 10.1042/bj1540243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leyten R., Vroemen J. P., Blanckaert N., Heirwegh K. P. The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation. Lab Anim. 1986 Oct;20(4):335–342. doi: 10.1258/002367786780808758. [DOI] [PubMed] [Google Scholar]
  17. Mackenzie P. I. Rat liver UDP-glucuronosyltransferase. Sequence and expression of a cDNA encoding a phenobarbital-inducible form. J Biol Chem. 1986 May 5;261(13):6119–6125. [PubMed] [Google Scholar]
  18. Meissner G., Allen R. Evidence for two types of rat liver microsomes with differing permeability to glucose and other small molecules. J Biol Chem. 1981 Jun 25;256(12):6413–6422. [PubMed] [Google Scholar]
  19. Nilsson R., Peterson E., Dallner G. Permeability of microsomal membranes isolated from rat liver. J Cell Biol. 1973 Mar;56(3):762–776. doi: 10.1083/jcb.56.3.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Perez M., Hirschberg C. B. Topography of glycosylation reactions in the rough endoplasmic reticulum membrane. J Biol Chem. 1986 May 25;261(15):6822–6830. [PubMed] [Google Scholar]
  21. Perez M., Hirschberg C. B. Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus. J Biol Chem. 1985 Apr 25;260(8):4671–4678. [PubMed] [Google Scholar]
  22. Ravoet A. M., Amar-Costesec A., Godelaine D., Beaufay H. Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver. J Cell Biol. 1981 Dec;91(3 Pt 1):679–688. doi: 10.1083/jcb.91.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scher M. G., Burton W. A., Waechter C. J. Enzymatic glucosylation of dolichyl monophosphate formed via cytidine triphosphate in calf brain membranes. J Neurochem. 1980 Oct;35(4):844–849. doi: 10.1111/j.1471-4159.1980.tb07081.x. [DOI] [PubMed] [Google Scholar]
  24. Sommers L. W., Hirschberg C. B. Transport of sugar nucleotides into rat liver Golgi. A new Golgi marker activity. J Biol Chem. 1982 Sep 25;257(18):10811–10817. [PubMed] [Google Scholar]
  25. Vanstapel F., Blanckaert N. Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase. J Biol Chem. 1987 Apr 5;262(10):4616–4623. [PubMed] [Google Scholar]
  26. Vanstapel F., Pua K., Blanckaert N. Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations. Eur J Biochem. 1986 Apr 1;156(1):73–77. doi: 10.1111/j.1432-1033.1986.tb09550.x. [DOI] [PubMed] [Google Scholar]

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