Skip to main content
SpringerLink
Log in

Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage

  • Protocol
  • First Online:
BCL-2 Family Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1877))

Abstract

Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Czabotar PE, Lessene G, Strasser A, Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15(1):49–63. https://doi.org/10.1038/nrm3722

    Article  CAS  PubMed  Google Scholar 

  2. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family reunion. Mol Cell 37(3):299–310. https://doi.org/10.1016/j.molcel.2010.01.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dai H, Smith A, Meng XW, Schneider PA, Pang YP, Kaufmann SH (2011) Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization. J Cell Biol 194(1):39–48. https://doi.org/10.1083/jcb.201102027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Moldoveanu T, Grace CR, Llambi F, Nourse A, Fitzgerald P, Gehring K, Kriwacki RW, Green DR (2013) BID-induced structural changes in BAK promote apoptosis. Nat Struct Mol Biol 20(5):589–597. https://doi.org/10.1038/nsmb.2563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD (2010) BH3-Triggered Structural reorganization drives the activation of proapoptotic BAX. Mol Cell 40(3):481–492. https://doi.org/10.1016/j.molcel.2010.10.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Uren RT, Iyer S, Kluck RM (2017) Pore formation by dimeric Bak and Bax: an unusual pore? Philos Trans R Soc Lond Ser B Biol Sci 372(1726). https://doi.org/10.1098/rstb.2016.0218

    Article  CAS  Google Scholar 

  7. Alsop AE, Fennell SC, Bartolo RC, Tan IK, Dewson G, Kluck RM (2015) Dissociation of Bak alpha1 helix from the core and latch domains is required for apoptosis. Nat Commun 6:6841. https://doi.org/10.1038/ncomms7841

    Article  CAS  PubMed  Google Scholar 

  8. Czabotar PE, Westphal D, Dewson G, Ma S, Hockings C, Fairlie WD, Lee EF, Yao S, Robin AY, Smith BJ, Huang DC, Kluck RM, Adams JM, Colman PM (2013) Bax crystal structures reveal how BH3 domains activate bax and nucleate its oligomerization to induce apoptosis. Cell 152(3):519–531. https://doi.org/10.1016/j.cell.2012.12.031

    Article  CAS  PubMed  Google Scholar 

  9. Brouwer JM, Westphal D, Dewson G, Robin AY, Uren RT, Bartolo R, Thompson GV, Colman PM, Kluck RM, Czabotar PE (2014) Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers. Mol Cell 55(6):938–946. https://doi.org/10.1016/j.molcel.2014.07.016

    Article  CAS  PubMed  Google Scholar 

  10. Dewson G, Kratina T, Sim HW, Puthalakath H, Adams JM, Colman PM, Kluck RM (2008) To trigger apoptosis Bak exposes its BH3 domain and homo-dimerizes via BH3:grooove interactions. Mol Cell 30(3):369–380

    Article  CAS  PubMed  Google Scholar 

  11. Dewson G, Ma S, Frederick P, Hockings C, Tan I, Kratina T, Kluck RM (2012) Bax dimerizes via a symmetric BH3:groove interface during apoptosis. Cell Death Differ 19(4):661–670. https://doi.org/10.1038/cdd.2011.138

    Article  CAS  PubMed  Google Scholar 

  12. Uren RT, O'Hely M, Iyer S, Bartolo R, Shi MX, Brouwer JM, Alsop AE, Dewson G, Kluck RM (2017) Disordered clusters of Bak dimers rupture mitochondria during apoptosis. elife 6:e19944. https://doi.org/10.7554/eLife.19944

    Article  PubMed  PubMed Central  Google Scholar 

  13. Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, Garcia-Saez AJ, Bordignon E (2014) Structural model of active bax at the membrane. Mol Cell 56(4):496–505. https://doi.org/10.1016/j.molcel.2014.09.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Walensky LD, Gavathiotis E (2011) BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore. Trends Biochem Sci 36(12):642–652. https://doi.org/10.1016/j.tibs.2011.08.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kluck RM, Esposti MD, Perkins G, Renken C, Kuwana T, Bossy-Wetzel E, Goldberg M, Allen T, Barber MJ, Green DR, Newmeyer DD (1999) The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol 147(4):809–822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Uren RT, Dewson G, Bonzon C, Lithgow T, Newmeyer DD, Kluck RM (2005) Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes. J Biol Chem 280(3):2266–2274

    Article  CAS  PubMed  Google Scholar 

  17. PMSF (2006) Cold Spring Harbor Laboratory Press. http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8772.short

  18. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB, Korsmeyer SJ (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14(16):2060–2071

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL, Dillon CP, Green DR (2011) A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol Cell 44(4):517–531. https://doi.org/10.1016/j.molcel.2011.10.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bleicken S, Classen M, Padmavathi PV, Ishikawa T, Zeth K, Steinhoff HJ, Bordignon E (2010) Molecular details of Bax activation, oligomerization, and membrane insertion. J Biol Chem 285(9):6636–6647. https://doi.org/10.1074/jbc.M109.081539

    Article  CAS  PubMed  Google Scholar 

  21. Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, Korsmeyer SJ, Shore GC (1998) Regulated targeting of BAX to mitochondria. J Cell Biol 143(1):207–215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dewson G (2016) Characterizing Bcl-2 family protein conformation and oligomerization using cross-linking and antibody gel-shift in conjunction with native PAGE. Methods Mol Biol 1419:185–196. https://doi.org/10.1007/978-1-4939-3581-9_14

    Article  PubMed  Google Scholar 

  23. Dewson G, Kratina T, Czabotar P, Day CL, Adams JM, Kluck RM (2009) Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices. Mol Cell 36(4):696–703. https://doi.org/10.1016/j.molcel.2009.11.008

    Article  CAS  PubMed  Google Scholar 

  24. Iyer S, Anwari K, Alsop AE, Yuen WS, Huang DC, Carroll J, Smith NA, Smith BJ, Dewson G, Kluck RM (2016) Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies. Nat Commun 7:11734. https://doi.org/10.1038/ncomms11734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Iyer S, Bell F, Westphal D, Anwari K, Gulbis J, Smith BJ, Dewson G, Kluck RM (2015) Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains. Cell Death Differ. https://doi.org/10.1038/cdd.2015.15

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported by NHMRC project grants 637337 and 1008434 and program grant 1016701, the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS.

Author information

Authors and Affiliations

  1. Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

    Sweta Iyer, Rachel T. Uren & Ruth M. Kluck

  2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia

    Sweta Iyer, Rachel T. Uren & Ruth M. Kluck

Authors
  1. Sweta Iyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rachel T. Uren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ruth M. Kluck
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruth M. Kluck .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Evripidis Gavathiotis

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Iyer, S., Uren, R.T., Kluck, R.M. (2019). Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage. In: Gavathiotis, E. (eds) BCL-2 Family Proteins. Methods in Molecular Biology, vol 1877. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8861-7_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8861-7_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8860-0

  • Online ISBN: 978-1-4939-8861-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation