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Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein.

ABSTRACT: In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required.

SUBMITTER: Roucou X

PROVIDER: S-EPMC1223025 | biostudies-other | 2002 Dec

REPOSITORIES: biostudies-other

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BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane.

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Project description:The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E, Bid. Finally, Bid-null MEFs were substantially more resistant to apoptosis induced by heat shock, which has been shown to be dependent on apical activation of Caspase-2. The data are consistent with a model in which Caspase-2 induces apoptosis via cleavage of Bid at D59 and the subsequent engagement of the mitochondrial (intrinsic) pathway.

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