, Volume 15, Issue 5, pp 553–562 | Cite as

Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane

  • Vidyaramanan Ganesan
  • Meenu N. Perera
  • David Colombini
  • Debra Datskovskiy
  • Kirti Chadha
  • Marco Colombini
Original Paper


A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.


Apoptosis Mitochondria Ceramide Channel Bcl-2 Protein release 

Supplementary material

10495_2009_449_MOESM1_ESM.tif (19.2 mb)
Fig. S1: LaCl3 was added to an activated Bax-enhanced ceramide channel (2 μg ceramide and 11 nM activated Bax). Disassembly occurred after 1.8 min. The experiment shown is representative of more than 8 experiments. Amounts of added ceramide and ac-Bax were different between experiments. (TIFF 19,699 kb)
10495_2009_449_MOESM2_ESM.tif (12.5 mb)
Fig. S2: SDS–PAGE showing purity of recombinant Bax preparation. (TIFF 12,806 kb)
10495_2009_449_MOESM3_ESM.tif (3.5 mb)
Fig. S3: SDS–PAGE showing restricted digestion of activated Bax by trypsin. The digested fragment is 15 kDa as expected for detergent activated Bax. (TIFF 3,577 kb)


  1. 1.
    Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9:139–150CrossRefPubMedGoogle Scholar
  2. 2.
    Pettus BJ, Chalfant CE, Hannun YA (2002) Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta 1585:114–125PubMedGoogle Scholar
  3. 3.
    Siskind LJ, Kolesnick RN, Colombini M (2002) Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins. J Biol Chem 277:26796–26803CrossRefPubMedGoogle Scholar
  4. 4.
    Siskind LJ, Feinstein L, Yu T, Davis JS, Jones D et al (2008) Anti-apoptotic Bcl-2 family proteins disassemble ceramide channels. J Biol Chem 283:6622–6630CrossRefPubMedGoogle Scholar
  5. 5.
    Charles AG, Han TY, Liu YY, Hansen N, Giuliano AE, Cabot MC (2001) Taxol-induced ceramide generation and apoptosis in human breast cancer cells. Cancer Chemother Pharmacol 47:444–450CrossRefPubMedGoogle Scholar
  6. 6.
    Kroesen BJ, Pettus B, Luberto C, Busman M, Sietsma H et al (2001) Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J Biol Chem 276:13606–13614PubMedGoogle Scholar
  7. 7.
    Rodriguez-Lafrasse C, Alphonse G, Broquet P, Aloy MT, Louisot P (2001) Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis. Biochem J 357:407–416CrossRefPubMedGoogle Scholar
  8. 8.
    Thomas RL, Matsko CM, Lotze MT, Amoscato AA (1999) Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J Biol Chem 274:30580–30588CrossRefPubMedGoogle Scholar
  9. 9.
    Ardail D, Maalouf M, Boivin A, Chapet O, Bodennec J (2009) Diversity and complexity of ceramide generation after exposure of jurkat leukemia cells to irradiation. Int J Radiat Oncol Biol Phys 73:1211–1218PubMedGoogle Scholar
  10. 10.
    Birbes H, Luberto C, Hsu YT, El Bawab S, Hannun YA et al (2005) A mitochondrial pool of sphingomyelin is involved in TNFalpha-induced Bax translocation to mitochondria. Biochem J 386:445–451CrossRefPubMedGoogle Scholar
  11. 11.
    Dai Q, Liu J, Chen J, Durrant D, McIntyre TM et al (2004) Mitochondrial ceramide increases in UV-irradiated HeLa cells and is mainly derived from hydrolysis of sphingomyelin. Oncogene 23:3650–3658CrossRefPubMedGoogle Scholar
  12. 12.
    Matsko CM, Hunter OC, Rabinowich H, Lotze MT, Amoscato AA (2001) Mitochondrial lipid alterations during Fas- and radiation-induced apoptosis. Biochem Biophys Res Commun 287:1112–1120CrossRefPubMedGoogle Scholar
  13. 13.
    Vance JE (1990) Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem 265:7248–7256PubMedGoogle Scholar
  14. 14.
    Siskind LJ, Kolesnick RN, Colombini M (2006) Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations. Mitochondrion 6:118–125CrossRefPubMedGoogle Scholar
  15. 15.
    Zhou L, Chang D (2006) Dynamics and structure of the Bax–Bak complex responsible for releasing mitochondrial proteins during apoptosis. J Cell Sci 121:2186–2196CrossRefGoogle Scholar
  16. 16.
    Roucou X, Rostovtseva T, Monessuit S, Martinou JC, Antonsson B (2002) Bid induces cytochrome c-impermeable Bax channels in liposomes. Biochem J 363:547–552CrossRefPubMedGoogle Scholar
  17. 17.
    Guihard G, Bellot G, Moreau C, Pradal G, Ferry N, Thomy R et al (2004) The mitochondrial apoptosis-induced channel (MAC) corresponds to a late apoptotic event. J Biol Chem 279:46542–46550CrossRefPubMedGoogle Scholar
  18. 18.
    Antonsson B (2004) Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem 256–257:141–155CrossRefPubMedGoogle Scholar
  19. 19.
    Schlesinger PH, Gross A, Yin XM, Yamamoto K, Saito M et al (1997) Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA 94:11357–11362CrossRefPubMedGoogle Scholar
  20. 20.
    Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S et al (1997) Inhibition of Bax channel-forming activity by Bcl-2. Science 277:370–372CrossRefPubMedGoogle Scholar
  21. 21.
    Yang Z, Khoury C, Jean-Baptise G, Greenwood MT (2006) Identification of mouse sphingomyelin synthase 1 as a suppressor of Bax-mediated cell death in yeast. FEMS Yeast Res 6:751–762CrossRefPubMedGoogle Scholar
  22. 22.
    Neise D, Graupner V, Gillissen BF, Daniel PT, Schulze-Osthoff K et al (2008) Activation of the mitochondrial death pathway is commonly mediated by a preferential engagement of Bak. Oncogene 27:1387–1396CrossRefPubMedGoogle Scholar
  23. 23.
    von Haefen C, Wieder T, Gillissen B, Stärck L, Graupner V et al (2002) Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells. Oncogene 21:4009–4019CrossRefGoogle Scholar
  24. 24.
    Kashar H, Weigmann K, Yazdanpanah B, Haubert D, Kronke M (2005) Acid sphingomyelinase is indispensable for UV light induced-Bax conformational change at the mitochondrial membrane. J Biol Chem 280:20804–20813CrossRefGoogle Scholar
  25. 25.
    Lee AC, Xu X, Blachly-Dyson E, Forte M, Colombini M (1998) The role of yeast VDAC genes on the permeability of the mitochondrial outer membrane. J Membr Biol 161:173–181CrossRefPubMedGoogle Scholar
  26. 26.
    Suzuki M, Youle RJ, Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103:645–654CrossRefPubMedGoogle Scholar
  27. 27.
    Lucken-Ardjomande S, Montessuit S, Martinou JC (2008) Bax activation and stress induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes. Cell Death Differ 15:484–493CrossRefPubMedGoogle Scholar
  28. 28.
    Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M et al (2000) Bid, Bax and lipids cooperate to form supramolecular openings in the mitochondrial outer membrane. Cell 111:331–342CrossRefGoogle Scholar
  29. 29.
    Colombini M (1987) Characterization of channels isolated from plant mitochondria. Methods Enzymol 148:465–475CrossRefPubMedGoogle Scholar
  30. 30.
    Kluck RM, Esposti MD, Perkins G, Renken C, Kuwana T et al (1999) The pro-apoptotic proteins Bid and Bax cause limited permeabilization of mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol 147:809–822CrossRefPubMedGoogle Scholar
  31. 31.
    Ivashyna O, Garcia-Saez AJ, Ries J, Christenson ET, Schwille P et al (2009) Detergent-activated Bax protein is a monomer. J Biol Chem 284:23935–23946CrossRefPubMedGoogle Scholar
  32. 32.
    Siskind LJ, Colombini M (2000) The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis. J Biol Chem 275:38640–38644CrossRefPubMedGoogle Scholar
  33. 33.
    Di Paola M, Zaccagnino P, Montedoro G, Cocco T, Lorusso M (2004) Ceramide induces release of pro-apoptotic proteins from mitochondria by either a Ca2+ dependent or a Ca2+ independent mechanism. J Bioenerg Biomembr 36:165–170CrossRefPubMedGoogle Scholar
  34. 34.
    Birbes H, El Bawab S, Hannun YA, Obeid LM (2000) Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis. FASEB J 15:2669–2679CrossRefGoogle Scholar
  35. 35.
    Siskind LJ (2005) Mitochondrial ceramide and induction of apoptosis. J Bioenerg Biomembr 37:143–153CrossRefPubMedGoogle Scholar
  36. 36.
    Bionda C, Portoukalian J, Schmitt D, Rodriguez-Lafrasse C, Ardail D (2004) Subcellular compartmentation of ceramide metabolism: MAM (mitochondria associated membrane) and/or mitochondria? Biochem J 382:527–533CrossRefPubMedGoogle Scholar
  37. 37.
    Siskind LJ, Fluss S, Bui M, Colombini M (2005) Sphingosine forms channels in membranes that differ greatly from those formed from ceramide. J Bioenerg Biomembr 37:227–236CrossRefPubMedGoogle Scholar
  38. 38.
    Jurgensmeier JM, Zie Z, Deveraux Q, Ellerby L, Bredesen D et al (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95:4997–5002CrossRefPubMedGoogle Scholar
  39. 39.
    Munoz-Pinedo C, Guio-Carrion A, Goldstein JC, Fitzgerald P, Newmeyer DD et al (2006) Different intermembrane space proteins are released from mitochondria in a manner that is coordinately initiated but can vary in duration. Proc Natl Acad Sci USA 103:11573–11578CrossRefPubMedGoogle Scholar
  40. 40.
    Hsu YT, Youle RJ (1997) Non-ionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 272:13829–13834CrossRefPubMedGoogle Scholar
  41. 41.
    Sawada M, Nakashima S, Banno Y, Yamakawa H, Takenaka K et al (2000) Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells. Oncogene 19:3508–3520CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Vidyaramanan Ganesan
    • 1
  • Meenu N. Perera
    • 2
  • David Colombini
    • 2
  • Debra Datskovskiy
    • 2
  • Kirti Chadha
    • 2
  • Marco Colombini
    • 1
    • 2
  1. 1.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA
  2. 2.Department of BiologyUniversity of MarylandCollege ParkUSA

Personalised recommendations