Advertisement

Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol

  • Joachim Lauterwasser
  • Franziska Fimm-Todt
  • Frank Edlich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

BCL-2 proteins control stress-dependent commitment to the programmed cell death apoptosis. In nonapoptotic cells the proapoptotic BCL-2 proteins BAX and BAK but also prosurvival family members, like BCL-xL or MCL-1, translocate to the outer mitochondrial membrane (OMM) and retrotranslocate from the mitochondria back into the cytosol. The resulting equilibrium produces a broad range of localization pattern observed for BAX and BAK in human cells and shows correlation between relative BAX and BAK localizations and cellular predisposition to apoptosis. The retrotranslocation of BCL-2 proteins from the OMM can be measured using fluorescence-labeled protein in intact cells or endogenous protein from isolated heavy membrane fractions.

Key words

BCL-2 proteins Mitochondria Apoptosis Retrotranslocation BH3-only proteins 

References

  1. 1.
    Tait SWG, Green DR (2010) Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11:621–632.  https://doi.org/10.1038/nrm2952CrossRefGoogle Scholar
  2. 2.
    Martinou J-C, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 21:92–101.  https://doi.org/10.1016/j.devcel.2011.06.017CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, Neutzner A, Tjandra N, Youle RJ (2011) Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. Cell 145:104–116.  https://doi.org/10.1016/j.cell.2011.02.034CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Todt F, Cakir Z, Reichenbach F, Youle RJ, Edlich F (2013) The C-terminal helix of Bcl-x(L) mediates Bax retrotranslocation from the mitochondria. Cell Death Differ 20:333–342.  https://doi.org/10.1038/cdd.2012.131CrossRefPubMedGoogle Scholar
  5. 5.
    Todt F, Cakir Z, Reichenbach F, Emschermann F, Lauterwasser J, Kaiser A, Ichim G, Tait SWG, Frank S, Langer HF, Edlich F (2015) Differential retrotranslocation of mitochondrial Bax and Bak. EMBO J 34:67–80.  https://doi.org/10.15252/embj.201488806CrossRefPubMedGoogle Scholar
  6. 6.
    Cheng EHY, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ (2003) VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301:513–517.  https://doi.org/10.1126/science.1083995CrossRefPubMedGoogle Scholar
  7. 7.
    Lauterwasser J, Todt F, Zerbes RM, Nguyen TN, Craigen W, Lazarou M, van der Laan M, Edlich F (2016) The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation. Sci Rep 6:32994.  https://doi.org/10.1038/srep32994CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lazarou M, Stojanovski D, Frazier AE, Kotevski A, Dewson G, Craigen WJ, Kluck RM, Vaux DL, Ryan MT (2010) Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor. J Biol Chem 285:36876–36883.  https://doi.org/10.1074/jbc.M110.159301CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ma SB, Nguyen TN, Tan I, Ninnis R, Iyer S, Stroud DA, Menard M, Kluck RM, Ryan MT, Dewson G (2014) Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function. Cell Death Differ 21:1925–1935.  https://doi.org/10.1038/cdd.2014.119CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cakir Z, Lauterwasser J, Todt F, Funk K, Zerbes RM, Tanaka A, van der Laan M, Edlich F (2017) Parkin promotes proteasomal degradation of misregulated Bax. J Cell Sci 130(17):2903–2913CrossRefPubMedGoogle Scholar
  11. 11.
    Schellenberg B, Wang P, Keeble JA, Rodriguez-Enriquez R, Walker S, Owens TW, Foster F, Tanianis-Hughes J, Brennan K, Streuli CH, Gilmore AP (2013) Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming. Mol Cell 49:959–971.  https://doi.org/10.1016/j.molcel.2012.12.022CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Edlich F (2015) The great migration of Bax and Bak. Mol Cell Oncol 2:e995029.  https://doi.org/10.4161/23723556.2014.995029CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Reichenbach F, Wiedenmann C, Schalk E, Becker D, Funk K, Scholz-Kreisel P, Todt F, Wolleschak D, Dohner K, Marquardt JU, Heidel F, Edlich F (2017) Mitochondrial BAX determines the predisposition to apoptosis in human AML. Clin Cancer Res.  https://doi.org/10.1158/1078-0432.CCR-16-1941CrossRefPubMedGoogle Scholar
  14. 14.
    Ishikawa-Ankerhold HC, Ankerhold R, Drummen GPC (2012) Advanced fluorescence microscopy techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17:4047–4132.  https://doi.org/10.3390/molecules17044047CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Joachim Lauterwasser
    • 1
  • Franziska Fimm-Todt
    • 1
  • Frank Edlich
    • 1
    • 2
  1. 1.Faculty of Medicine, Institute of Biochemistry and Molecular Biology, ZBMZUniversity of FreiburgFreiburgGermany
  2. 2.BIOSS, Centre for Biological Signaling StudiesUniversity of FreiburgFreiburgGermany

Personalised recommendations