The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites

We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBidG94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBidG94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBidG94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.Recent work has shown that the Bcl-2 family regulates mitochondrial homeostasis during apoptosis [1]. Pro-apoptotic members, including Bax, Bak, Bid, and Bim, promote the release of death-inducing proteins, such as cytochrome c [2,3], smac [4], and endonuclease G [5], from mitochondria while anti-apoptotic members, such as Bcl-2 and Bcl-XL, inhibit this release. Following release into the cytosol, these death-inducing proteins promote apoptotic cell destruction through multiple pathways including caspase activation and nuclear DNA fragmentation.In addition to controlling the release of pro-death proteins, the Bcl-2 family also alters the function of mitochondria undergoing apoptosis. Dysfunction of vo