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. 2004 Feb 1;377(Pt 3):561–568. doi: 10.1042/BJ20031016

Ganglioside glycosyltransferases and newly synthesized gangliosides are excluded from detergent-insoluble complexes of Golgi membranes.

Pilar M Crespo 1, Adolfo R Zurita 1, Claudio G Giraudo 1, Hugo J F Maccioni 1, Jose L Daniotti 1
PMCID: PMC1223901  PMID: 14565845

Abstract

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

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Selected References

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

  1. Bagnat M., Keränen S., Shevchenko A., Shevchenko A., Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3254–3259. doi: 10.1073/pnas.060034697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bieberich Erhard, MacKinnon Sarah, Silva Jeane, Li Donna D., Tencomnao Tewin, Irwin Louis, Kapitonov Dmitri, Yu Robert K. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. Biochemistry. 2002 Sep 24;41(38):11479–11487. doi: 10.1021/bi0259958. [DOI] [PubMed] [Google Scholar]
  3. Bretscher M. S., Munro S. Cholesterol and the Golgi apparatus. Science. 1993 Sep 3;261(5126):1280–1281. doi: 10.1126/science.8362242. [DOI] [PubMed] [Google Scholar]
  4. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  5. Brügger B., Nickel W., Weber T., Parlati F., McNew J. A., Rothman J. E., Söllner T. Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors. EMBO J. 2000 Mar 15;19(6):1272–1278. doi: 10.1093/emboj/19.6.1272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crespo Pilar Maria, Zurita Adolfo Ramón, Daniotti Jose Luis. Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells. J Biol Chem. 2002 Sep 16;277(47):44731–44739. doi: 10.1074/jbc.M204604200. [DOI] [PubMed] [Google Scholar]
  7. Daniotti J. L., Martina J. A., Giraudo C. G., Zurita A. R., Maccioni H. J. GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells. J Neurochem. 2000 Apr;74(4):1711–1720. doi: 10.1046/j.1471-4159.2000.0741711.x. [DOI] [PubMed] [Google Scholar]
  8. Daniotti J. L., Martina J. A., Zurita A. R., Maccioni H. J. Mouse beta 1,3-galactosyltransferase (GA1/GM1/GD1b synthase): protein characterization, tissue expression, and developmental regulation in neural retina. J Neurosci Res. 1999 Oct 15;58(2):318–327. [PubMed] [Google Scholar]
  9. Dietrich C., Volovyk Z. N., Levi M., Thompson N. L., Jacobson K. Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers. Proc Natl Acad Sci U S A. 2001 Sep 4;98(19):10642–10647. doi: 10.1073/pnas.191168698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FOLCH J., ARSOVE S., MEATH J. A. Isolation of brain strandin, a new type of large molecule tissue component. J Biol Chem. 1951 Aug;191(2):819–831. [PubMed] [Google Scholar]
  11. Galbiati F., Razani B., Lisanti M. P. Emerging themes in lipid rafts and caveolae. Cell. 2001 Aug 24;106(4):403–411. doi: 10.1016/s0092-8674(01)00472-x. [DOI] [PubMed] [Google Scholar]
  12. Giraudo C. G., Daniotti J. L., Maccioni H. J. Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus. Proc Natl Acad Sci U S A. 2001 Jan 23;98(4):1625–1630. doi: 10.1073/pnas.031458398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Giraudo C. G., Rosales Fritz V. M., Maccioni H. J. GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells. Biochem J. 1999 Sep 15;342(Pt 3):633–640. [PMC free article] [PubMed] [Google Scholar]
  14. Giraudo Claudio G., Maccioni Hugo J. F. Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1. Mol Biol Cell. 2003 May 18;14(9):3753–3766. doi: 10.1091/mbc.E03-02-0101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giraudo Claudio G., Maccioni Hugo J. F. Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells. J Biol Chem. 2003 Aug 4;278(41):40262–40271. doi: 10.1074/jbc.M305455200. [DOI] [PubMed] [Google Scholar]
  16. Gkantiragas I., Brügger B., Stüven E., Kaloyanova D., Li X. Y., Löhr K., Lottspeich F., Wieland F. T., Helms J. B. Sphingomyelin-enriched microdomains at the Golgi complex. Mol Biol Cell. 2001 Jun;12(6):1819–1833. doi: 10.1091/mbc.12.6.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hakomori S. Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J Biol Chem. 1990 Nov 5;265(31):18713–18716. [PubMed] [Google Scholar]
  18. Hakomori S., Yamamura S., Handa A. K. Signal transduction through glyco(sphingo)lipids. Introduction and recent studies on glyco(sphingo)lipid-enriched microdomains. Ann N Y Acad Sci. 1998 Jun 19;845:1–10. doi: 10.1111/j.1749-6632.1998.tb09657.x. [DOI] [PubMed] [Google Scholar]
  19. Heino S., Lusa S., Somerharju P., Ehnholm C., Olkkonen V. M., Ikonen E. Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface. Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8375–8380. doi: 10.1073/pnas.140218797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ikonen E. Roles of lipid rafts in membrane transport. Curr Opin Cell Biol. 2001 Aug;13(4):470–477. doi: 10.1016/s0955-0674(00)00238-6. [DOI] [PubMed] [Google Scholar]
  21. Iwabuchi K., Yamamura S., Prinetti A., Handa K., Hakomori S. GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J Biol Chem. 1998 Apr 10;273(15):9130–9138. doi: 10.1074/jbc.273.15.9130. [DOI] [PubMed] [Google Scholar]
  22. Kasahara K., Watanabe K., Takeuchi K., Kaneko H., Oohira A., Yamamoto T., Sanai Y. Involvement of gangliosides in glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid rafts. J Biol Chem. 2000 Nov 3;275(44):34701–34709. doi: 10.1074/jbc.M003163200. [DOI] [PubMed] [Google Scholar]
  23. Kasahara K., Watanabe Y., Yamamoto T., Sanai Y. Association of Src family tyrosine kinase Lyn with ganglioside GD3 in rat brain. Possible regulation of Lyn by glycosphingolipid in caveolae-like domains. J Biol Chem. 1997 Nov 21;272(47):29947–29953. doi: 10.1074/jbc.272.47.29947. [DOI] [PubMed] [Google Scholar]
  24. Keller P., Toomre D., Díaz E., White J., Simons K. Multicolour imaging of post-Golgi sorting and trafficking in live cells. Nat Cell Biol. 2001 Feb;3(2):140–149. doi: 10.1038/35055042. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Madore N., Smith K. L., Graham C. H., Jen A., Brady K., Hall S., Morris R. Functionally different GPI proteins are organized in different domains on the neuronal surface. EMBO J. 1999 Dec 15;18(24):6917–6926. doi: 10.1093/emboj/18.24.6917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maxzúd M. K., Daniotti J. L., Maccioni H. J. Functional coupling of glycosyl transfer steps for synthesis of gangliosides in Golgi membranes from neural retina cells. J Biol Chem. 1995 Aug 25;270(34):20207–20214. doi: 10.1074/jbc.270.34.20207. [DOI] [PubMed] [Google Scholar]
  28. Nichols B. J., Kenworthy A. K., Polishchuk R. S., Lodge R., Roberts T. H., Hirschberg K., Phair R. D., Lippincott-Schwartz J. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol. 2001 Apr 30;153(3):529–541. doi: 10.1083/jcb.153.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Orlandi P. A., Fishman P. H. Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J Cell Biol. 1998 May 18;141(4):905–915. doi: 10.1083/jcb.141.4.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pelham H. R., Rothman J. E. The debate about transport in the Golgi--two sides of the same coin? Cell. 2000 Sep 15;102(6):713–719. doi: 10.1016/s0092-8674(00)00060-x. [DOI] [PubMed] [Google Scholar]
  31. Prinetti A., Iwabuchi K., Hakomori S. Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis. J Biol Chem. 1999 Jul 23;274(30):20916–20924. doi: 10.1074/jbc.274.30.20916. [DOI] [PubMed] [Google Scholar]
  32. Rosales Fritz V. M., Daniotti J. L., Maccioni H. J. Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase. Biochim Biophys Acta. 1997 Nov 1;1354(2):153–158. doi: 10.1016/s0167-4781(97)00117-6. [DOI] [PubMed] [Google Scholar]
  33. SVENNERHOLM L. CHROMATOGRAPHIC SEPARATION OF HUMAN BRAIN GANGLIOSIDES. J Neurochem. 1963 Sep;10:613–623. doi: 10.1111/j.1471-4159.1963.tb08933.x. [DOI] [PubMed] [Google Scholar]
  34. Sevlever D., Pickett S., Mann K. J., Sambamurti K., Medof M. E., Rosenberry T. L. Glycosylphosphatidylinositol-anchor intermediates associate with triton-insoluble membranes in subcellular compartments that include the endoplasmic reticulum. Biochem J. 1999 Nov 1;343(Pt 3):627–635. [PMC free article] [PubMed] [Google Scholar]
  35. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  36. Simons K., Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 2000 Oct;1(1):31–39. doi: 10.1038/35036052. [DOI] [PubMed] [Google Scholar]
  37. Simons K., van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988 Aug 23;27(17):6197–6202. doi: 10.1021/bi00417a001. [DOI] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vyas K. A., Patel H. V., Vyas A. A., Schnaar R. L. Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers. Biol Chem. 2001 Feb;382(2):241–250. doi: 10.1515/BC.2001.031. [DOI] [PubMed] [Google Scholar]
  40. Yamashita Tadashi, Hashiramoto Akira, Haluzik Martin, Mizukami Hiroki, Beck Shoshannah, Norton Aaron, Kono Mari, Tsuji Shuichi, Daniotti Jose Luis, Werth Norbert. Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S A. 2003 Mar 10;100(6):3445–3449. doi: 10.1073/pnas.0635898100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zurita A. R., Maccioni H. J., Daniotti J. L. Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides. Biochem J. 2001 Apr 15;355(Pt 2):465–472. doi: 10.1042/0264-6021:3550465. [DOI] [PMC free article] [PubMed] [Google Scholar]

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