Analysis of Proapoptotic Protein Trafficking to and from Mitochondria.

Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.