The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the "multidomain" proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halpha1 and their BH3 domains allows Bid and PUMA to function as "death agonists" of Bax, whereas Bad recruits Bax activity through a distinct pathway.

The translocation of Bax ␣, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program. We report here that the N-terminal (NT) end of Bax ␣, which contains its first ␣ helix (⌯␣1), is a functional mitochondrial-addressing signal both in mammals and in yeast. Similar results were obtained with a newly described variant of Bax called Bax , which lacks the first 20 amino acids of Bax ␣ and is constitutively associated with mitochondria. Deletion of ⌯␣1 impairs the binding of Bax to mitochondria, whereas a fusion of the N terminus of Bax ␣, which contains ⌯␣1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro. More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax with mitochondria as well as Baxapoptogenic properties. The mutations of the ⌯␣1, which inhibit Bax ␣ and Bax translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis. Conversely, a deletion of the C terminus does not appear to influence Bax ␣ and Bax mitochondrial addressing. Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family.Apoptosis is finely regulated by intracellular events, which at its onset, appear to be under the control of members of the BCL-2 family (1). The main site of action of these proteins appears to be the mitochondrion, particularly through the control of the release of apoptogenic factors from this organelle (2, 3). Members of the BCL-2 family can be anti-apoptotic or proapoptotic and totally or partially mitochondrial-bound or cytosolic (4). In most cells, one of the crucial and most regulated steps in the implementation of apoptosis is the control of the translocation of Bax ␣ from the cytosol to the mitochondria (4). Anti-apoptotic proteins such as Bcl-2 or Bcl-XL act as inhibitors of Bax function, whereas proapoptotic BH3 only members can either hinder this inhibition (e. g. Bad) or directly activate Bax ␣ (e. g. Bid), which in both cases would promote the association of Bax ␣ with the mitochondrial outer membrane (MOM) 1 (5, 6). Bcl-2 is anchored into MOM bilayer by a short hydrophobic domain close to the C terminus (CT) with its N terminus (NT) exposed toward the cytosol (7-9). Bcl-2 belongs to a class of membrane proteins called tail-anchored (TA) proteins, which are associated with different intracellular membranes, usually MOM and the endoplasmic reticulum (10). Based on the homology with Bcl-2, it has been proposed that Bax became inserted to mitochondria by its hydrophobic ␣9 helix located at its Cterminal end (for example, see Ref. 11). Several studies have shown that the Bax ␣ CT is not required for its interaction with mitochondria, whereas others have found it mandatory for Bax ␣ function (12-16). We have observed that a chimeric Bcl-XL construct in which the natural Bcl-XL CT was substituted by t...

The translocation of Bax from the cytosol into the mitochondrial outer membrane is a central event during apoptosis. We report that beyond the addressing step, which involves its first alpha-helix (halpha1), the helices alpha5 and alpha6 (halpha5alpha6) are responsible for the insertion of Bax into mitochondrial outer membrane bilayer. The translocation of Bax to mitochondria is associated with specific changes in the conformation of the protein that are under the control of two prolines: Pro-13, which controls the unfolding of halpha1, and Pro-168, a proline located immediately before the hydrophobic carboxyl-terminal end (i.e. helix alpha9, halpha9), which controls the disclosure of halpha5alpha6. An additional step, the disruption of an electrostatic bond formed between Asp-33 (halpha1) and Lys-64 (BH3), allows the mitochondria addressing of Bax. We conclude that, although the intramolecular interactions of halpha1 with the BH3 region control the addressing of Bax to mitochondria, the Pro-168 is involved in the control of its membrane insertion through halpha5alpha6.

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak ؊ BeGBM and Bak ؊ BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak ؊ BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak ؊ BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.