title: Identification of p53 in mitochondria.
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.
authors.affiliation: Department of Pathology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Angelina V
authors.initials: AV
authors.lastname: Vaseva
authors.forename: Ute M
authors.initials: UM
authors.lastname: Moll
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002448:Cell Adhesion; D002458:Cell Fractionation / Q000379:methods*; D004249:DNA Damage; D006801:Humans; D008928:Mitochondria / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D016159:Tumor Suppressor Protein p53