Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival full-length Bcl-x(L) and pro-death Bcl-x(L) cleavage products (Delta N612Bcl-x(L) and Delta N76Bcl-x(L)). Unlike Bcl-x(L), Delta N61Bcl-x(L) and Delta N76Bcl-x(L) caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant Delta N61Bcl-x(L) and Delta N76Bcl-x(L), as well as Bcl-xL, cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only Delta N61Bcl-x(L) and Delta N76Bcl-x(L), but not Bcl-x(L), formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x(L) and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor protein-protein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x(L) cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.