Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9:139–150

    Article  PubMed  CAS  Google Scholar 

  2. Pettus BJ, Chalfant CE, Hannun YA (2002) Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta 1585:114–125

    PubMed  CAS  Google Scholar 

  3. Siskind LJ, Kolesnick RN, Colombini M (2002) Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins. J Biol Chem 277:26796–26803

    Article  PubMed  CAS  Google Scholar 

  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 283:6622–6630

    Article  PubMed  CAS  Google Scholar 

  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 47:444–450

    Article  PubMed  CAS  Google Scholar 

  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 276:13606–13614

    PubMed  CAS  Google Scholar 

  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 357:407–416

    Article  PubMed  CAS  Google Scholar 

  8. Thomas RL, Matsko CM, Lotze MT, Amoscato AA (1999) Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J Biol Chem 274:30580–30588

    Article  PubMed  CAS  Google Scholar 

  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 73:1211–1218

    PubMed  CAS  Google Scholar 

  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 386:445–451

    Article  PubMed  CAS  Google Scholar 

  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 23:3650–3658

    Article  PubMed  CAS  Google Scholar 

  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 287:1112–1120

    Article  PubMed  CAS  Google Scholar 

  13. Vance JE (1990) Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem 265:7248–7256

    PubMed  CAS  Google Scholar 

  14. Siskind LJ, Kolesnick RN, Colombini M (2006) Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations. Mitochondrion 6:118–125

    Article  PubMed  CAS  Google Scholar 

  15. Zhou L, Chang D (2006) Dynamics and structure of the Bax–Bak complex responsible for releasing mitochondrial proteins during apoptosis. J Cell Sci 121:2186–2196

    Article  CAS  Google Scholar 

  16. Roucou X, Rostovtseva T, Monessuit S, Martinou JC, Antonsson B (2002) Bid induces cytochrome c-impermeable Bax channels in liposomes. Biochem J 363:547–552

    Article  PubMed  CAS  Google Scholar 

  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 279:46542–46550

    Article  PubMed  CAS  Google Scholar 

  18. Antonsson B (2004) Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem 256–257:141–155

    Article  PubMed  Google Scholar 

  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 94:11357–11362

    Article  PubMed  CAS  Google Scholar 

  20. Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S et al (1997) Inhibition of Bax channel-forming activity by Bcl-2. Science 277:370–372

    Article  PubMed  CAS  Google Scholar 

  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 6:751–762

    Article  PubMed  CAS  Google Scholar 

  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 27:1387–1396

    Article  PubMed  CAS  Google Scholar 

  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 21:4009–4019

    Article  CAS  Google Scholar 

  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 280:20804–20813

    Article  CAS  Google Scholar 

  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 161:173–181

    Article  PubMed  CAS  Google Scholar 

  26. Suzuki M, Youle RJ, Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103:645–654

    Article  PubMed  CAS  Google Scholar 

  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 15:484–493

    Article  PubMed  CAS  Google Scholar 

  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 111:331–342

    Article  Google Scholar 

  29. Colombini M (1987) Characterization of channels isolated from plant mitochondria. Methods Enzymol 148:465–475

    Article  PubMed  CAS  Google Scholar 

  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 147:809–822

    Article  PubMed  CAS  Google Scholar 

  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 284:23935–23946

    Article  PubMed  CAS  Google Scholar 

  32. Siskind LJ, Colombini M (2000) The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis. J Biol Chem 275:38640–38644

    Article  PubMed  CAS  Google Scholar 

  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 36:165–170

    Article  PubMed  CAS  Google Scholar 

  34. Birbes H, El Bawab S, Hannun YA, Obeid LM (2000) Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis. FASEB J 15:2669–2679

    Article  Google Scholar 

  35. Siskind LJ (2005) Mitochondrial ceramide and induction of apoptosis. J Bioenerg Biomembr 37:143–153

    Article  PubMed  CAS  Google Scholar 

  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 382:527–533

    Article  PubMed  CAS  Google Scholar 

  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 37:227–236

    Article  PubMed  CAS  Google Scholar 

  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 95:4997–5002

    Article  PubMed  CAS  Google Scholar 

  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 103:11573–11578

    Article  PubMed  CAS  Google Scholar 

  40. Hsu YT, Youle RJ (1997) Non-ionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 272:13829–13834

    Article  PubMed  CAS  Google Scholar 

  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 19:3508–3520

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the National Science Foundation (MCB-0641208). We are deeply in debt to Antonella Antignani and Richard Youle both for providing us with the plasmid we used to express full-length Bax and for assistance with the Bax isolation and purification procedure. We extend our gratitude to Don Newmeyer for the N/C Bid plasmid and Ryan Hastie for assistance with the purification procedure. We are very grateful to the following students who assisted with experiments on planar membranes: Raksha Bangalore, Dipkumar Patel, Kevin Yang and Chiemezie Onyewuchi. Finally, we wish to express our gratitude to Leah Siskind for her foundational work and advice during these studies.

Author information

Authors and Affiliations

  1. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA

    Vidyaramanan Ganesan & Marco Colombini

  2. Department of Biology, University of Maryland, Bldg 144, College Park, MD, 20742, USA

    Meenu N. Perera, David Colombini, Debra Datskovskiy, Kirti Chadha & Marco Colombini

Authors
  1. Vidyaramanan Ganesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meenu N. Perera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Colombini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Debra Datskovskiy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kirti Chadha
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marco Colombini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marco Colombini.

Additional information

Vidyaramanan Ganesan and Meenu N. Perera contributed equally to the work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10495_2009_449_MOESM1_ESM.tif

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)

Fig. S2: SDS–PAGE showing purity of recombinant Bax preparation. (TIFF 12,806 kb)

10495_2009_449_MOESM3_ESM.tif

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ganesan, V., Perera, M.N., Colombini, D. et al. Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane. Apoptosis 15, 553–562 (2010). https://doi.org/10.1007/s10495-009-0449-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-009-0449-0

Keywords

Search

Navigation