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. 1996 Apr 1;133(1):15–28. doi: 10.1083/jcb.133.1.15

Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis

PMCID: PMC2120776  PMID: 8601603

Abstract

Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used short-chain ceramides with either an 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA- and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (Ga2Cer). Although the HFA-GalCer synthesizing activity colocalized with an ER marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GalCer are synthesized in the cytosolic leaflet, while LacCer and Ga2Cer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GalCer, and HFA-GalCer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.

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Selected References

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  1. Ahnert-Hilger G., Mach W., Föhr K. J., Gratzl M. Poration by alpha-toxin and streptolysin O: an approach to analyze intracellular processes. Methods Cell Biol. 1989;31:63–90. doi: 10.1016/s0091-679x(08)61602-7. [DOI] [PubMed] [Google Scholar]
  2. Arion W. J. Measurement of intactness of rat liver endoplasmic reticulum. Methods Enzymol. 1989;174:58–67. doi: 10.1016/0076-6879(89)74010-6. [DOI] [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Babia T., Kok J. W., van der Haar M., Kalicharan R., Hoekstra D. Transport of biosynthetic sphingolipids from Golgi to plasma membrane in HT29 cells: involvement of different carrier vesicle populations. Eur J Cell Biol. 1994 Apr;63(2):172–181. [PubMed] [Google Scholar]
  5. Backer J. M., Dawidowicz E. A. Reconstitution of a phospholipid flippase from rat liver microsomes. 1987 May 28-Jun 3Nature. 327(6120):341–343. doi: 10.1038/327341a0. [DOI] [PubMed] [Google Scholar]
  6. Bansal R., Pfeiffer S. E. Regulated galactolipid synthesis and cell surface expression in Schwann cell line D6P2T. J Neurochem. 1987 Dec;49(6):1902–1911. doi: 10.1111/j.1471-4159.1987.tb02453.x. [DOI] [PubMed] [Google Scholar]
  7. Barondes S. H., Cooper D. N., Gitt M. A., Leffler H. Galectins. Structure and function of a large family of animal lectins. J Biol Chem. 1994 Aug 19;269(33):20807–20810. [PubMed] [Google Scholar]
  8. Bishop W. R., Bell R. M. Assembly of phospholipids into cellular membranes: biosynthesis, transmembrane movement and intracellular translocation. Annu Rev Cell Biol. 1988;4:579–610. doi: 10.1146/annurev.cb.04.110188.003051. [DOI] [PubMed] [Google Scholar]
  9. Bonner W. M., Stedman J. D. Efficient fluorography of 3H and 14C on thin layers. Anal Biochem. 1978 Aug 15;89(1):247–256. doi: 10.1016/0003-2697(78)90747-9. [DOI] [PubMed] [Google Scholar]
  10. Bossuyt X., Blanckaert N. Carrier-mediated transport of intact UDP-glucuronic acid into the lumen of endoplasmic-reticulum-derived vesicles from rat liver. Biochem J. 1994 Aug 15;302(Pt 1):261–269. doi: 10.1042/bj3020261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Briles E. B., Li E., Kornfeld S. Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J Biol Chem. 1977 Feb 10;252(3):1107–1116. [PubMed] [Google Scholar]
  12. Brändli A. W., Hansson G. C., Rodriguez-Boulan E., Simons K. A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex. J Biol Chem. 1988 Nov 5;263(31):16283–16290. [PubMed] [Google Scholar]
  13. Coates S. W., Gurney T., Jr, Sommers L. W., Yeh M., Hirschberg C. B. Subcellular localization of sugar nucleotide synthetases. J Biol Chem. 1980 Oct 10;255(19):9225–9229. [PubMed] [Google Scholar]
  14. Connor J., Pak C. H., Zwaal R. F., Schroit A. J. Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process. J Biol Chem. 1992 Sep 25;267(27):19412–19417. [PubMed] [Google Scholar]
  15. Coste H., Martel M. B., Got R. Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands. Biochim Biophys Acta. 1986 Jun 13;858(1):6–12. doi: 10.1016/0005-2736(86)90285-3. [DOI] [PubMed] [Google Scholar]
  16. Deutscher S. L., Hirschberg C. B. Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes. J Biol Chem. 1986 Jan 5;261(1):96–100. [PubMed] [Google Scholar]
  17. Deutscher S. L., Nuwayhid N., Stanley P., Briles E. I., Hirschberg C. B. Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport. Cell. 1984 Dec;39(2 Pt 1):295–299. doi: 10.1016/0092-8674(84)90007-2. [DOI] [PubMed] [Google Scholar]
  18. Drake R. R., Igari Y., Lester R., Elbein A. D., Radominska A. Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases. J Biol Chem. 1992 Jun 5;267(16):11360–11365. [PubMed] [Google Scholar]
  19. Farrer R. G., Warden M. P., Quarles R. H. Effects of brefeldin A on galactosphingolipid synthesis in an immortalized Schwann cell line: evidence for different intracellular locations of galactosylceramide sulfotransferase and ceramide galactosyltransferase activities. J Neurochem. 1995 Oct;65(4):1865–1873. doi: 10.1046/j.1471-4159.1995.65041865.x. [DOI] [PubMed] [Google Scholar]
  20. Futerman A. H., Pagano R. E. Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver. Biochem J. 1991 Dec 1;280(Pt 2):295–302. doi: 10.1042/bj2800295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gillard B. K., Thurmon L. T., Marcus D. M. Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells. Cell Motil Cytoskeleton. 1992;21(4):255–271. doi: 10.1002/cm.970210402. [DOI] [PubMed] [Google Scholar]
  22. Hammond C., Helenius A. Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus. J Cell Biol. 1994 Jul;126(1):41–52. doi: 10.1083/jcb.126.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hansson G. C., Simons K., van Meer G. Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions. EMBO J. 1986 Mar;5(3):483–489. doi: 10.1002/j.1460-2075.1986.tb04237.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Herrmann A., Zachowski A., Devaux P. F. Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity. Biochemistry. 1990 Feb 27;29(8):2023–2027. doi: 10.1021/bi00460a010. [DOI] [PubMed] [Google Scholar]
  25. Hirschberg C. B., Snider M. D. Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem. 1987;56:63–87. doi: 10.1146/annurev.bi.56.070187.000431. [DOI] [PubMed] [Google Scholar]
  26. Hirschberg K., Rodger J., Futerman A. H. The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver. Biochem J. 1993 Mar 15;290(Pt 3):751–757. doi: 10.1042/bj2900751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Inokuchi J., Radin N. S. Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase. J Lipid Res. 1987 May;28(5):565–571. [PubMed] [Google Scholar]
  28. Jeckel D., Karrenbauer A., Burger K. N., van Meer G., Wieland F. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions. J Cell Biol. 1992 Apr;117(2):259–267. doi: 10.1083/jcb.117.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kean E. L. Separation of gluco- and galactocerebrosides by means of borate thin-layer chromatography. J Lipid Res. 1966 May;7(3):449–452. [PubMed] [Google Scholar]
  30. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kotani M., Hosoya H., Kubo H., Itoh K., Sakuraba H., Kusubata M., Inagaki M., Yazaki S., Suzuki Y., Tai T. Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts. Cell Struct Funct. 1994 Apr;19(2):81–87. doi: 10.1247/csf.19.81. [DOI] [PubMed] [Google Scholar]
  32. Lannert H., Bünning C., Jeckel D., Wieland F. T. Lactosylceramide is synthesized in the lumen of the Golgi apparatus. FEBS Lett. 1994 Mar 28;342(1):91–96. doi: 10.1016/0014-5793(94)80591-1. [DOI] [PubMed] [Google Scholar]
  33. Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4132–4136. doi: 10.1073/pnas.77.7.4132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mandon E. C., Ehses I., Rother J., van Echten G., Sandhoff K. Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver. J Biol Chem. 1992 Jun 5;267(16):11144–11148. [PubMed] [Google Scholar]
  35. Mayer L. D., Hope M. J., Cullis P. R. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta. 1986 Jun 13;858(1):161–168. doi: 10.1016/0005-2736(86)90302-0. [DOI] [PubMed] [Google Scholar]
  36. McIntyre J. C., Sleight R. G. Fluorescence assay for phospholipid membrane asymmetry. Biochemistry. 1991 Dec 24;30(51):11819–11827. doi: 10.1021/bi00115a012. [DOI] [PubMed] [Google Scholar]
  37. Morell P., Radin N. S. Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid. Biochemistry. 1969 Feb;8(2):506–512. doi: 10.1021/bi00830a008. [DOI] [PubMed] [Google Scholar]
  38. Nilsson T., Warren G. Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curr Opin Cell Biol. 1994 Aug;6(4):517–521. doi: 10.1016/0955-0674(94)90070-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nimura Y., Isihizuka I. Glycosphingolipid composition of a renal cell line (MDCK) and its ouabain-resistant mutant. J Biochem. 1986 Oct;100(4):825–835. doi: 10.1093/oxfordjournals.jbchem.a121794. [DOI] [PubMed] [Google Scholar]
  40. Pagano R. E., Martin O. C. A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells. Biochemistry. 1988 Jun 14;27(12):4439–4445. doi: 10.1021/bi00412a034. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Rijnboutt S., Aerts H. M., Geuze H. J., Tager J. M., Strous G. J. Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells. J Biol Chem. 1991 Mar 15;266(8):4862–4868. [PubMed] [Google Scholar]
  43. Sasaki T., Demel R. A. Net mass transfer of galactosylceramide facilitated by glycolipid transfer protein from pig brain: a monolayer study. Biochemistry. 1985 Feb 26;24(5):1079–1083. doi: 10.1021/bi00326a002. [DOI] [PubMed] [Google Scholar]
  44. Sasaki T. Glycolipid transfer protein and intracellular traffic of glucosylceramide. Experientia. 1990 Jun 15;46(6):611–616. doi: 10.1007/BF01939700. [DOI] [PubMed] [Google Scholar]
  45. Sato C., Black J. A., Yu R. K. Subcellular distribution of UDP-galactose:ceramide galactosyltransferase in rat brain oligodendroglia. J Neurochem. 1988 Jun;50(6):1887–1893. doi: 10.1111/j.1471-4159.1988.tb02493.x. [DOI] [PubMed] [Google Scholar]
  46. Schaeren-Wiemers N., van der Bijl P., Schwab M. E. The UDP-galactose:ceramide galactosyltransferase: expression pattern in oligodendrocytes and Schwann cells during myelination and substrate preference for hydroxyceramide. J Neurochem. 1995 Nov;65(5):2267–2278. doi: 10.1046/j.1471-4159.1995.65052267.x. [DOI] [PubMed] [Google Scholar]
  47. Schulte S., Stoffel W. Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10265–10269. doi: 10.1073/pnas.90.21.10265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schweizer A., Clausen H., van Meer G., Hauri H. P. Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment. J Biol Chem. 1994 Feb 11;269(6):4035–4041. [PubMed] [Google Scholar]
  49. Spitalnik P. F., Danley J. M., Burger S. R., Spitalnik S. L. The glycosphingolipid composition of the human hepatoma cell line,Hep-G2. Arch Biochem Biophys. 1989 Sep;273(2):578–591. doi: 10.1016/0003-9861(89)90518-3. [DOI] [PubMed] [Google Scholar]
  50. Stahl N., Jurevics H., Morell P., Suzuki K., Popko B. Isolation, characterization, and expression of cDNA clones that encode rat UDP-galactose: ceramide galactosyltransferase. J Neurosci Res. 1994 Jun 1;38(2):234–242. doi: 10.1002/jnr.490380214. [DOI] [PubMed] [Google Scholar]
  51. Stanley P., Siminovitch L. Complementation between mutants of CHO cells resistant to a variety of plant lectins. Somatic Cell Genet. 1977 Jul;3(4):391–405. doi: 10.1007/BF01542968. [DOI] [PubMed] [Google Scholar]
  52. Strous G. J., van Kerkhof P., van Meer G., Rijnboutt S., Stoorvogel W. Differential effects of brefeldin A on transport of secretory and lysosomal proteins. J Biol Chem. 1993 Feb 5;268(4):2341–2347. [PubMed] [Google Scholar]
  53. Tennekoon G., Zaruba M., Wolinsky J. Topography of cerebroside sulfotransferase in Golgi-enriched vesicles from rat brain. J Cell Biol. 1983 Oct;97(4):1107–1112. doi: 10.1083/jcb.97.4.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Trinchera M., Fabbri M., Ghidoni R. Topography of glycosyltransferases involved in the initial glycosylations of gangliosides. J Biol Chem. 1991 Nov 5;266(31):20907–20912. [PubMed] [Google Scholar]
  55. Trinchera M., Fiorilli A., Ghidoni R. Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase. Biochemistry. 1991 Mar 12;30(10):2719–2724. doi: 10.1021/bi00224a021. [DOI] [PubMed] [Google Scholar]
  56. Vunnam R. R., Radin N. S. Short chain ceramides as substrates for glucocerebroside synthetase. Differences between liver and brain enzymes. Biochim Biophys Acta. 1979 Apr 27;573(1):73–82. doi: 10.1016/0005-2760(79)90174-7. [DOI] [PubMed] [Google Scholar]
  57. Warnock D. E., Lutz M. S., Blackburn W. A., Young W. W., Jr, Baenziger J. U. Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2708–2712. doi: 10.1073/pnas.91.7.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Willem J., ter Beest M., Scherphof G., Hoekstra D. A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway. Eur J Cell Biol. 1990 Oct;53(1):173–184. [PubMed] [Google Scholar]
  59. Yusuf H. K., Pohlentz G., Sandhoff K. Tunicamycin inhibits ganglioside biosynthesis in rat liver Golgi apparatus by blocking sugar nucleotide transport across the membrane vesicles. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7075–7079. doi: 10.1073/pnas.80.23.7075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zilversmit D. B., Hughes M. E. Extensive exchange of rat liver microsomal phospholipids. Biochim Biophys Acta. 1977 Aug 15;469(1):99–110. doi: 10.1016/0005-2736(77)90329-7. [DOI] [PubMed] [Google Scholar]
  61. van Echten G., Sandhoff K. Ganglioside metabolism. Enzymology, Topology, and regulation. J Biol Chem. 1993 Mar 15;268(8):5341–5344. [PubMed] [Google Scholar]
  62. van Genderen I. L., van Meer G., Slot J. W., Geuze H. J., Voorhout W. F. Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution. J Cell Biol. 1991 Nov;115(4):1009–1019. doi: 10.1083/jcb.115.4.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. van Meer G., Burger K. N. Sphingolipid trafficking--sorted out? Trends Cell Biol. 1992 Nov;2(11):332–337. [PubMed] [Google Scholar]
  64. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. van den Besselaar A. M., de Druijff B., van den Bosch H., van Deenen L. L. Phosphatidylcholine mobility in liver microsomal membranes. Biochim Biophys Acta. 1978 Jul 4;510(2):242–255. doi: 10.1016/0005-2736(78)90024-x. [DOI] [PubMed] [Google Scholar]

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