The C-terminal helix of Bcl-x(L) mediates Bax retrotranslocation from the mitochondria

19.10.2012

Todt F, Cakir Z, Reichenbach F, Youle RJ, Edlich F.

Cell Death Differ. 2013 Feb;20(2):333-42

Cell Death Differ.          online article

The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-x(L), control Bax activity. Bcl-x(L) recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-x(L). Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-x(L) membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-x(L), but not that of Bcl-x(L) shuttling. Bax retrotranslocation depends on interaction to the Bcl-x(L) membrane anchor and interaction between the Bax BH3 domain and the Bcl-x(L) hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction.

The pro-apoptotic Bcl-2 protein Bax can commit cells to apoptosis. Bax activity is controlled in healthy cells by the pro-survival Bcl-xL protein (a). Bcl-xL (white circle) recognizes Bax (grey square) on the outer mitochondrial membrane (OMM) and retrotranslocates Bax into the cytoplasm stabilizing the inactive form of Bax. If Bax retrotranslocation is impaired by interfering with the interaction of the C-terminal membrane anchor of Bcl-xL with Bax (?C, H9) or with the recognition of the Bax BH3 by Bcl-xL (ABT-737) Bax accumulates on the OMM (b, c). Similar mitochondrial Bax levels can be detected on the OMM after apoptosis stimulation (c, STS), but in the absence of apoptotic stimuli mitochondrial Bax is not inducing apoptosis. However, the size of the mitochondrial Bax pool directly correlates to the apoptotic cell fate after apoptosis stimulation.