Identification of p53 in mitochondria

Angelina V. Vaseva, Ute M. Moll

Producción científica: Chapter

14 Citas (Scopus)

Resumen

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.

Idioma originalEnglish (US)
Título de la publicación alojadap53 Protocols
EditorialHumana Press
Páginas75-84
Número de páginas10
ISBN (versión impresa)9781627032353
DOI
EstadoPublished - 2013
Publicado de forma externa

Serie de la publicación

NombreMethods in Molecular Biology
Volumen962
ISSN (versión impresa)1064-3745

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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