Mitochondria were known to play a central role in apoptosis. Specifically, releases of cytochrome c (Cyt c) and Smac from mitochondria are the key steps that trigger the activation of caspase cascades during cell death. At present, it is still unclear how the release of mitochondrial proteins is regulated by the Bcl-2/Bax family proteins. To solve this problem, we have used GFP-gene fusion and living cell image techniques to study: (1) The translocation of Bax from the cytosol to mitochondria during UV-induced apoptosis, (2) The temporal relationship between Bax translocation and the releases of Cyt c and Smac from mitochondria. We observed that the distribution of Bax underwent four phases of distribution during UV-induced apoptosis. The releases of Cyt c and Smac occurred at the same...[ Read more ]

Mitochondria were known to play a central role in apoptosis. Specifically, releases of cytochrome c (Cyt c) and Smac from mitochondria are the key steps that trigger the activation of caspase cascades during cell death. At present, it is still unclear how the release of mitochondrial proteins is regulated by the Bcl-2/Bax family proteins. To solve this problem, we have used GFP-gene fusion and living cell image techniques to study: (1) The translocation of Bax from the cytosol to mitochondria during UV-induced apoptosis, (2) The temporal relationship between Bax translocation and the releases of Cyt c and Smac from mitochondria. We observed that the distribution of Bax underwent four phases of distribution during UV-induced apoptosis. The releases of Cyt c and Smac occurred at the same time as the third phase of Bax distribution, i.e., formation of Bax small aggregates. Fluorescence resonance energy transfer (FRET) study further revealed that the formation of small aggregates coincided with Bax and Bak interaction. These small aggregates were composed by thousands of molecules. We conclude that lipid-protein complex formed in the outer mitochondrial membrane by Bax and Bak aggregates during apoptosis is most likely responsible for the release of mitochondrial intermembrane proteins.

At present, there is a strong interest to identify the functional domains that are correlated with Bax translocation, aggregation and pro-apoptotic function. We fused YFP with various Bax deletion mutants, in which at least one of the four putative functional domains of Bax was deleted: the ART domain, the BH3 domain, the α5/6 helices and the TM domain. Single cell analysis of these Bax deletion mutants demonstrated that: (1) ART at the N-terminus appeared to function as an inhibitory domain for Bax activation. (2) TM at the C-terminus of Bax was responsible for mitochondrial targeting during apoptosis. (3) The BH3 domain was essential for the pro-apoptotic function of Bax by forming small aggregates in mitochondria. (4) The α5/6 helices appeared to be responsible for Bax cluster formation during apoptosis. Taken together, our results suggest that, upon an apoptotic induction, the targeting of Bax to mitochondria, aggregation, triggering of apoptosis, and cluster formation are separately mediated by discrete domains of Bax.

Finally, we investigated how Bax is activated during apoptosis. Several hypotheses have been suggested in the literature, including: (1) Removing the inhibitory binding proteins of Bax (e.g. Mcl-1, Bcl-2, Bcl-xL, 14-3-3 or Ku70); (2) Bax binding with some BH3-only proteins (e.g. tBid, Bim); (3) other mechanisms, such as up-regulation and N-terminus cleavage of Bax. Our findings have excluded several of these hypotheses for Bax activation, including tBid or Bad binding, up-regulation and N-terminus cleavage of Bax. On the other hand, our results suggest that the elimination of Mcl-1 and JNK-mediated Bim activation are probably the major causes for Bax activation.

In conclusion, our studies support a new model for Bax activation and Bax-mediated mitochondria-dependent apoptotic pathway: In normal conditions, Bax is sequestered by some inhibitory proteins in the cytosol and mitochondria. Upon apoptotic stimuli, these inhibitory proteins are inactivated and Bax is released. Concurrently, some activated BH3 only proteins can bind with Bax through the BH3 domain, causing a conformational change of Bax and translocation to mitochondria, where Bax/Bak form protein-lipid complexes to permeabilize the mitochondrial outer membrane.