Identification of p53 in mitochondria

Angelina V. Vaseva, Ute M. Moll

Research output: Chapter in Book/Report/Conference proceedingChapter

12 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationp53 Protocols
PublisherHumana Press
Pages75-84
Number of pages10
ISBN (Print)9781627032353
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume962
ISSN (Print)1064-3745

Keywords

  • Mitochondria purification
  • p53
  • Submitochondrial fractionation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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