Identification of p53 in Mitochondria

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 962)

Abstract

p53 is a master regulator of cell death pathways and has transcription-dependent and transcription-independent modes of action. Mitochondria are major signal transducers in apoptosis and are critical for p53-dependent cell death. Our lab and others have discovered that a fraction of stress-induced wild-type p53 protein rapidly translocates to mitochondria upon various stress stimuli and exerts p53-dependent apoptosis. Suborganellar localization by various methods shows that p53 localizes to the surface of mitochondria. Direct targeting of p53 to mitochondria is sufficient to induce apoptosis in p53-null cells, without requiring further DNA damage. Recently, p53 has been also shown to localize to other mitochondrial compartments such as the mitochondrial matrix where it plays a role in maintaining mitochondrial genome integrity. Here, we describe subcellular fractionation as a classic technique for detecting mitochondrial p53 in cell extracts. It consists of cell homogenization by hypo-osmotic swelling, removal of nuclear components by low-speed centrifugation, and mitochondrial isolation by a discontinuous sucrose density gradient. Additionally, we describe a method for submitochondrial fractionation, performed by phosphate buffer mediated swelling/shrinking. p53 and other mitochondrial proteins can then be detected by standard immunoblotting procedures. The quality of mitochondrial isolates/subfractions can be verified for purity and intactness.

Key words

p53 Mitochondria purification Submitochondrial fractionation 

References

  1. 1.
    Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323–331PubMedCrossRefGoogle Scholar
  2. 2.
    Harris CC (1996) Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88:1442–1455PubMedCrossRefGoogle Scholar
  3. 3.
    Gottlieb TM, Oren M (1998) p53 and apoptosis. Semin Cancer Biol 8:359–368PubMedCrossRefGoogle Scholar
  4. 4.
    Brenner C, Kroemer G (2000) Apoptosis. Mitochondria – the death signal integrators. Science 289:1150–1151PubMedCrossRefGoogle Scholar
  5. 5.
    Moll UM, Zaika A (2001) Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett 493:65–69PubMedCrossRefGoogle Scholar
  6. 6.
    Marchenko ND, Zaika A, Moll UM (2000) Death signal-induced localization of p53 ­protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 275:16202–16212PubMedCrossRefGoogle Scholar
  7. 7.
    Sansome C, Zaika A, Marchenko ND, Moll UM (2001) Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells. FEBS Lett 488:110–115PubMedCrossRefGoogle Scholar
  8. 8.
    Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM (2003) p53 has a direct apoptogenic role at the mitochondria. Mol Cell 11:577–590PubMedCrossRefGoogle Scholar
  9. 9.
    Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR (2004) Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303:1010–1014PubMedCrossRefGoogle Scholar
  10. 10.
    Talos F, Petrenko O, Mena P, Moll UM (2005) Mitochondrially targeted p53 has tumor ­suppressor activities in vivo. Cancer Res 65:9971–9981PubMedCrossRefGoogle Scholar
  11. 11.
    Palacios G, Moll UM (2006) Mitochondrially targeted wild-type p53 suppresses growth of mutant p53 lymphomas in vivo. Oncogene 25:6133–6139PubMedCrossRefGoogle Scholar
  12. 12.
    Palacios G, Crawford HC, Vaseva A, Moll UM (2008) Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model. Cell Cycle 7(16):2584–2590PubMedCrossRefGoogle Scholar
  13. 13.
    Achanta G, Sasaki R, Feng L, Carew JS, Lu W, Pelicano H, Keating MJ, Huang P (2005) Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma. EMBO J 24:3482–3492PubMedCrossRefGoogle Scholar
  14. 14.
    de Souza-Pinto NC, Harris CC, Bohr VA (2004) p53 functions in the incorporation step in DNA base excision repair in mouse liver mitochondria. Oncogene 23:6559–6568PubMedCrossRefGoogle Scholar
  15. 15.
    Yoshida Y, Izumi H, Torigoe T, Ishiguchi H, Itoh H, Kang D, Kohno K (2003) P53 physically interacts with mitochondrial transcription factor A and differentially regulates binding to damaged DNA. Cancer Res 63:3729–3734PubMedGoogle Scholar
  16. 16.
    Vamecq J, Van Hoof F (1984) Implication of a peroxisomal enzyme in the catabolism of glutaryl-CoA. Biochem J 221:203–211PubMedGoogle Scholar
  17. 17.
    Bhattacharya SK, Thakar JH, Johnson PL, Shanklin DR (1991) Isolation of skeletal muscle mitochondria from hamsters using an ionic medium containing ethylenediaminetetraacetic acid and nagarse. Anal Biochem 192:344–349PubMedCrossRefGoogle Scholar
  18. 18.
    Beauvoit B, Rigoulet M, Guerin B (1989) Thermodynamic and kinetic control of ATP synthesis in yeast mitochondria: role of delta pH. FEBS Lett 244:255–258PubMedCrossRefGoogle Scholar
  19. 19.
    Bogenhagen D, Clayton DA (1974) The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J Biol Chem 249:7991–7995PubMedGoogle Scholar
  20. 20.
    Hovius R, Lambrechts H, Nicolay K, de Kruijff B (1990) Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim Biophys Acta 1021:217–226PubMedCrossRefGoogle Scholar
  21. 21.
    Bijur GN, Jope RS (2003) Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation. J Neurochem 87:1427–1435PubMedCrossRefGoogle Scholar
  22. 22.
    Milon L, Meyer P, Chiadmi M, Munier A, Johansson M, Karlsson A, Lascu I, Capeau J, Janin J, Lacombe ML (2000) The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase. J Biol Chem 275:14264–14272PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of PathologyStony Brook UniversityStony BrookUSA

Personalised recommendations