Document Detail

Identification of p53 in Mitochondria.
MedLine Citation:
PMID:  23150438     Owner:  NLM     Status:  Publisher    
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.
Angelina V Vaseva; Ute M Moll
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Publication Detail:
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  962     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-11-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  75-84     Citation Subset:  -    
Department of Pathology, Stony Brook University, Stony Brook, NY, USA.
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