The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites.
BACKGROUND
Following cleavage by caspase 8, the C-terminus of Bid translocates from the cytosol to the mitochondria that is dependent upon structures formed by the mitochondrial-specific lipid cardiolipin. Once associated with mitochondria, truncated Bid (tBid) causes the potent release of cytochrome c, endonuclease G, and smac.


RESULTS
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.


CONCLUSIONS
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.
Lutter
M
M
Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA. michael.lutter@utsouthwestern.edu
Perkins
G A
GA
Wang
X
X
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000737:chemistry* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D008928:Mitochondria / Q000737:chemistry*; D008930:Mitochondria, Liver / Q000737:chemistry / Q000648:ultrastructure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D014054:Tomography
D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2