Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 May 1;371(Pt 3):1013–1019. doi: 10.1042/BJ20021834

The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases.

Dominique Ardail 1, Iuliana Popa 1, Jacques Bodennec 1, Pierre Louisot 1, Daniel Schmitt 1, Jacques Portoukalian 1
PMCID: PMC1223353  PMID: 12578562

Abstract

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

Full Text

The Full Text of this article is available as a PDF (134.8 KB).

Selected References

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

  1. Achleitner G., Gaigg B., Krasser A., Kainersdorfer E., Kohlwein S. D., Perktold A., Zellnig G., Daum G. Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur J Biochem. 1999 Sep;264(2):545–553. doi: 10.1046/j.1432-1327.1999.00658.x. [DOI] [PubMed] [Google Scholar]
  2. Anand J. K., Sadozai K. K., Hakomori S. A simple method for the synthesis of ceramides and radiolabeled analogues. Lipids. 1996 Sep;31(9):995–998. doi: 10.1007/BF02522695. [DOI] [PubMed] [Google Scholar]
  3. Ardail D., Gasnier F., Lermé F., Simonot C., Louisot P., Gateau-Roesch O. Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes. J Biol Chem. 1993 Dec 5;268(34):25985–25992. [PubMed] [Google Scholar]
  4. Ardail D., Popa I., Alcantara K., Pons A., Zanetta J. P., Louisot P., Thomas L., Portoukalian J. Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria. FEBS Lett. 2001 Jan 19;488(3):160–164. doi: 10.1016/s0014-5793(00)02332-2. [DOI] [PubMed] [Google Scholar]
  5. Ardail D., Popa I., Bodennec J., Famy C., Louisot P., Portoukalian J. Subcellular distribution and metabolic fate of exogenous ceramides taken up by HL-60 cells. Biochim Biophys Acta. 2002 Aug 8;1583(3):305–310. doi: 10.1016/s1388-1981(02)00252-4. [DOI] [PubMed] [Google Scholar]
  6. Bodennec J., Koul O., Aguado I., Brichon G., Zwingelstein G., Portoukalian J. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J Lipid Res. 2000 Sep;41(9):1524–1531. [PubMed] [Google Scholar]
  7. Bouchon B., Portoukalian J., Bornet H. Sex-specific difference of the galabiosylceramide level in the glycosphingolipids of human thyroid. Biochim Biophys Acta. 1985 Aug 22;836(1):143–152. doi: 10.1016/0005-2760(85)90230-9. [DOI] [PubMed] [Google Scholar]
  8. Bouchon B., Portoukalian J., Orgiazzi J., Bornet H. Selective enrichment of phytosphingosine in glycosphingolipids of isolated human thyrocytes as compared to the whole thyroid. Biochem Biophys Res Commun. 1987 Mar 30;143(3):827–831. doi: 10.1016/0006-291x(87)90323-8. [DOI] [PubMed] [Google Scholar]
  9. Bouhours J. F., Guignard H. Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells. J Lipid Res. 1979 Sep;20(7):879–907. [PubMed] [Google Scholar]
  10. 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]
  11. Burger K. N., van der Bijl P., van Meer G. Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis. J Cell Biol. 1996 Apr;133(1):15–28. doi: 10.1083/jcb.133.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chandra N. C., Spiro M. J., Spiro R. G. Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum. J Biol Chem. 1998 Jul 31;273(31):19715–19721. doi: 10.1074/jbc.273.31.19715. [DOI] [PubMed] [Google Scholar]
  13. Croze E. M., Morré D. J. Isolation of plasma membrane, golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver. J Cell Physiol. 1984 Apr;119(1):46–57. doi: 10.1002/jcp.1041190109. [DOI] [PubMed] [Google Scholar]
  14. Daum G., Lees N. D., Bard M., Dickson R. Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast. 1998 Dec;14(16):1471–1510. doi: 10.1002/(SICI)1097-0061(199812)14:16<1471::AID-YEA353>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
  15. Dnistrian A. M., Skipski V. P., Barclay M., Stock C. C. Glycosphingolipids of subcellular fractions from normal rat liver and Morris hepatoma 5123TC. J Natl Cancer Inst. 1979 Feb;62(2):367–370. [PubMed] [Google Scholar]
  16. Funato K., Riezman H. Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast. J Cell Biol. 2001 Dec 3;155(6):949–959. doi: 10.1083/jcb.200105033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gillard B. K., Thurmon L. T., Marcus D. M. Variable subcellular localization of glycosphingolipids. Glycobiology. 1993 Feb;3(1):57–67. doi: 10.1093/glycob/3.1.57. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Keenan T. W., Huang C. M., Morré D. J. Gangliosides: nonspecific localization in the surface membranes of bovine mammary gland and rat liver. Biochem Biophys Res Commun. 1972 Jun 28;47(6):1277–1283. doi: 10.1016/0006-291x(72)90211-2. [DOI] [PubMed] [Google Scholar]
  20. Kendler A., Dawson G. Hypoxic injury to oligodendrocytes: reversible inhibition of ATP-dependent transport of ceramide from the endoplasmic reticulum to the Golgi. J Neurosci Res. 1992 Feb;31(2):205–211. doi: 10.1002/jnr.490310202. [DOI] [PubMed] [Google Scholar]
  21. Kok J. W., Babia T., Klappe K., Egea G., Hoekstra D. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated. Biochem J. 1998 Aug 1;333(Pt 3):779–786. doi: 10.1042/bj3330779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ladinsky M. S., Mastronarde D. N., McIntosh J. R., Howell K. E., Staehelin L. A. Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J Cell Biol. 1999 Mar 22;144(6):1135–1149. doi: 10.1083/jcb.144.6.1135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lester R. L., Dickson R. C. Sphingolipids with inositolphosphate-containing head groups. Adv Lipid Res. 1993;26:253–274. [PubMed] [Google Scholar]
  25. 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]
  26. Mao C., Xu R., Szulc Z. M., Bielawska A., Galadari S. H., Obeid L. M. Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide. J Biol Chem. 2001 May 16;276(28):26577–26588. doi: 10.1074/jbc.M102818200. [DOI] [PubMed] [Google Scholar]
  27. Marcus D. M., Schwarting G. A. Immunochemical properties of glycolipids and phospholipids. Adv Immunol. 1976;23:203–240. [PubMed] [Google Scholar]
  28. Matsuo N., Nomura T., Imokawa G. A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase. Biochim Biophys Acta. 1992 Apr 22;1116(2):97–103. doi: 10.1016/0304-4165(92)90105-4. [DOI] [PubMed] [Google Scholar]
  29. Matyas G. R., Morré D. J. Subcellular distribution and biosynthesis of rat liver gangliosides. Biochim Biophys Acta. 1987 Oct 17;921(3):599–614. doi: 10.1016/0005-2760(87)90089-0. [DOI] [PubMed] [Google Scholar]
  30. Merrill Alfred H., Jr De novo sphingolipid biosynthesis: a necessary, but dangerous, pathway. J Biol Chem. 2002 May 13;277(29):25843–25846. doi: 10.1074/jbc.R200009200. [DOI] [PubMed] [Google Scholar]
  31. Miller-Podraza H., Bradley R. M., Fishman P. H. Biosynthesis and localization of gangliosides in cultured cells. Biochemistry. 1982 Jul 6;21(14):3260–3265. doi: 10.1021/bi00257a002. [DOI] [PubMed] [Google Scholar]
  32. Moreau P., Cassagne C., Keenan T. W., Morré D. J. Ceramide excluded from cell-free vesicular lipid transfer from endoplasmic reticulum to Golgi apparatus. Evidence for lipid sorting. Biochim Biophys Acta. 1993 Feb 23;1146(1):9–16. doi: 10.1016/0005-2736(93)90332-t. [DOI] [PubMed] [Google Scholar]
  33. Popa Iuliana, Vlad Cristina, Bodennec Jacques, Portoukalian Jacques. Recovery of gangliosides from aqueous solutions on styrene-divinylbenzene copolymer columns. J Lipid Res. 2002 Aug;43(8):1335–1340. [PubMed] [Google Scholar]
  34. Puoti A., Desponds C., Conzelmann A. Biosynthesis of mannosylinositolphosphoceramide in Saccharomyces cerevisiae is dependent on genes controlling the flow of secretory vesicles from the endoplasmic reticulum to the Golgi. J Cell Biol. 1991 May;113(3):515–525. doi: 10.1083/jcb.113.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rubin M. S., Tzagoloff A. Cytochrome oxidase of Saccharomyces cerevisiae. Methods Enzymol. 1978;53:73–79. doi: 10.1016/s0076-6879(78)53015-2. [DOI] [PubMed] [Google Scholar]
  36. Rusiñol A. E., Cui Z., Chen M. H., Vance J. E. A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins. J Biol Chem. 1994 Nov 4;269(44):27494–27502. [PubMed] [Google Scholar]
  37. 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]
  38. Shiao Y. J., Balcerzak B., Vance J. E. A mitochondrial membrane protein is required for translocation of phosphatidylserine from mitochondria-associated membranes to mitochondria. Biochem J. 1998 Apr 1;331(Pt 1):217–223. doi: 10.1042/bj3310217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shiao Y. J., Lupo G., Vance J. E. Evidence that phosphatidylserine is imported into mitochondria via a mitochondria-associated membrane and that the majority of mitochondrial phosphatidylethanolamine is derived from decarboxylation of phosphatidylserine. J Biol Chem. 1995 May 12;270(19):11190–11198. doi: 10.1074/jbc.270.19.11190. [DOI] [PubMed] [Google Scholar]
  40. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  41. Trinchera M., Carrettoni D., Ghidoni R. A part of glucosylceramide formed from exogenous lactosylceramide is not degraded to ceramide but re-cycled and glycosylated in the Golgi apparatus. J Biol Chem. 1991 May 15;266(14):9093–9099. [PubMed] [Google Scholar]
  42. Vance J. E. Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem. 1990 May 5;265(13):7248–7256. [PubMed] [Google Scholar]
  43. Vance J. E., Vance D. E. Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J Biol Chem. 1988 Apr 25;263(12):5898–5909. [PubMed] [Google Scholar]
  44. Venkataraman K., Futerman A. H. Comparison of the metabolism of L-erythro- and L-threo-sphinganines and ceramides in cultured cells and in subcellular fractions. Biochim Biophys Acta. 2001 Feb 26;1530(2-3):219–226. doi: 10.1016/s1388-1981(01)00085-3. [DOI] [PubMed] [Google Scholar]
  45. Vidugiriene J., Sharma D. K., Smith T. K., Baumann N. A., Menon A. K. Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum. J Biol Chem. 1999 May 21;274(21):15203–15212. doi: 10.1074/jbc.274.21.15203. [DOI] [PubMed] [Google Scholar]
  46. Voelker D. R. The ATP-dependent translocation of phosphatidylserine to the mitochondria is a process that is restricted to the autologous organelle. J Biol Chem. 1993 Apr 5;268(10):7069–7074. [PubMed] [Google Scholar]
  47. 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]
  48. van Echten G., Sandhoff K. Ganglioside metabolism. Enzymology, Topology, and regulation. J Biol Chem. 1993 Mar 15;268(8):5341–5344. [PubMed] [Google Scholar]
  49. van Meer Gerrit, Lisman Quirine. Sphingolipid transport: rafts and translocators. J Biol Chem. 2002 May 13;277(29):25855–25858. doi: 10.1074/jbc.R200010200. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES