Skip to main content

Investigating BCL-2 Family Protein Interactions in Yeast

  • Protocol
  • First Online:

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

Abstract

Among the different models used to study the biochemical properties and function of proteins of the Bcl-2 family, their heterologous expression in the yeast Saccharomyces cerevisiae may look out of place. However, when grown under adequate conditions, yeast cells have mitochondria that have similar properties as those of mammalian cells, and are able to be targeted by mammalian Bcl-2 family members. Yeast thus provides a neutral cellular background to study how proteins of the Bcl-2 family interact with mitochondria, alone or as a couple (or a combination of more proteins). Most studies done in our laboratory has been done on the proapoptotic protein Bax and the antiapoptotic protein Bcl-xL, but yeast can bring about useful information about every protein of the family, in terms of their capacity to interact and to regulate the permeabilization of the outer mitochondrial membrane.

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

Buying options

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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. 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 152:519–531

    Article  CAS  PubMed  Google Scholar 

  2. Simonyan L, Légiot A, Lascu I, Durand G, Giraud MF, Gonzalez C, Manon S (2017) The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria. Biochim Biophys Acta 1859:1144–1155

    Article  CAS  Google Scholar 

  3. Antonsson B, Montessuit S, Lauper S, Eskes R, Martinou JC (2000) Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J 345:271–278

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Manon S, Chaudhuri B, Guérin M (1997) Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL. FEBS Lett 415:29–32

    Article  CAS  PubMed  Google Scholar 

  5. Jürgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A 95:4997–5002

    Article  PubMed Central  PubMed  Google Scholar 

  6. Carmona-Gutierrez D, Eisenberg T, Büttner S, Meisinger C, Kroemer G, Madeo F (2010) Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 17:763–773

    Article  CAS  PubMed  Google Scholar 

  7. Renault TT, Dejean LM, Manon S (2017) A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2. Mech Ageing Dev 161:201–210

    Article  CAS  PubMed  Google Scholar 

  8. Priault M, Cartron PF, Camougrand N, Antonsson B, Vallette FM, Manon S (2003) Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria. Cell Death Differ 10:1068–1077

    Article  CAS  PubMed  Google Scholar 

  9. Arokium H, Camougrand N, Vallette FM, Manon S (2004) Studies of the interaction of substituted mutants of BAX with yeast mitochondria reveal that the C-terminal hydrophobic alpha-helix is a second ART sequence and plays a role in the interaction with anti-apoptotic BCL-xL. J Biol Chem 279:52566–52573

    Article  CAS  PubMed  Google Scholar 

  10. Arokium H, Ouerfelli H, Velours G, Camougrand N, Vallette FM, Manon S (2007) Substitutions of potentially phosphorylatable serine residues of Bax reveal how they may regulate its interaction with mitochondria. J Biol Chem 282:35104–35112

    Article  CAS  PubMed  Google Scholar 

  11. Simonyan L, Renault TT, Novais MJ, Sousa MJ, Côrte-Real M, Camougrand N, Gonzalez C, Manon S (2016) Regulation of Bax/mitochondria interaction by AKT. FEBS Lett 590:13–21

    Article  CAS  PubMed  Google Scholar 

  12. Garenne D, Renault TT, Manon S (2016) Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL. Microb Cell 3:597–605

    Article  PubMed Central  PubMed  Google Scholar 

  13. Renault TT, Teijido O, Missire F, Ganesan YT, Velours G, Arokium H, Beaumatin F, Llanos R, Athané A, Camougrand N, Priault M, Antonsson B, Dejean LM, Manon S (2015) Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737. Int J Biochem Cell Biol 64:136–146

    Article  CAS  PubMed  Google Scholar 

  14. Gonzalvez F, Bessoule JJ, Rocchiccioli F, Manon S, Petit PX (2005) Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria. Cell Death Differ 12:659–667

    Article  CAS  PubMed  Google Scholar 

  15. Gallenne T, Gautier F, Oliver L, Hervouet E, Noël B, Hickman JA, Geneste O, Cartron PF, Vallette FM, Manon S, Juin P (2009) Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members. J Cell Biol 185:279–290

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Prudent J, Popgeorgiev N, Bonneau B, Thibaut J, Gadet R, Lopez J, Gonzalo P, Rimokh R, Manon S, Houart C, Herbomel P, Aouacheria A, Gillet G (2013) Bcl-wav and the mitochondrial calcium uniporter drive gastrula morphogenesis in zebrafish. Nat Commun 4:2330

    Article  PubMed  Google Scholar 

  17. Bellot G, Cartron PF, Er E, Oliver L, Juin P, Armstrong LC, Bornstein P, Mihara K, Manon S, Vallette FM (2007) TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax. Cell Death Differ 14:785–794

    Article  CAS  PubMed  Google Scholar 

  18. Renault TT, Grandier-Vazeille X, Arokium H, Velours G, Camougrand N, Priault M, Teijido O, Dejean LM, Manon S (2012) The cytosolic domain of human Tom22 modulates human Bax mitochondrial translocation and conformation in yeast. FEBS Lett 586:116–121

    Article  CAS  PubMed  Google Scholar 

  19. Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62:334–361

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Garí E, Piedrafita L, Aldea M, Herrero E (1997) A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13:837–848

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The work in the lab of the author is supported by the CNRS and the Université of Bordeaux. The author wishes to thank the PhD students who have optimized these protocols, namely Muriel Priault, Hubert Arokium, and Thibaud T. Renault.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stéphen Manon .

Editor information

Editors and Affiliations

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

Manon, S. (2019). Investigating BCL-2 Family Protein Interactions in Yeast. 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_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8861-7_6

  • 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