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Abstract
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 (DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L)). Unlike Bcl-x(L), DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L) caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), as well as Bcl-x(L), cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only DeltaN61Bcl-x(L) and DeltaN76Bcl-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.
Highlights
Proteins of the Bcl-2 family are key regulators of programmed cell death in multicellular organisms
Reaction mixtures were centrifuged at 14,000 ϫ g for 10 min, mitochondrial membrane pellets corresponding to 25 g of protein and the corresponding volume of supernatants were separated by SDS-PAGE on 4 –20% Tris-glycine gels, and their respective cytochrome c contents were estimated by immunoblotting (7H8.2C-12) using the ECL method (Amersham Pharmacia Biotech)
Pro-apoptotic Bcl-xL Cleavage Products Release Cytochrome c from Mitochondria—Because the pro-apoptotic activity of Bcl-2 family proteins appears to rely on their ability to release cytochrome c from mitochondria, we first studied the effect of the pro-apoptotic C-terminal portion of Bcl-xL on cytochrome c distribution in living cells
Summary
Vol 276, No 33, Issue of August 17, pp. 31083–31091, 2001 Printed in U.S.A. Pro-apoptotic Cleavage Products of Bcl-xL Form Cytochrome c-conducting Pores in Pure Lipid Membranes*. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c We tested this hypothesis using pro-survival fulllength Bcl-xL and pro-death Bcl-xL cleavage products (⌬N61Bcl-xL and ⌬N76Bcl-xL). Because Bcl-xL and its C-terminal cleavage products bound to lipid membranes and formed oligomers of the same size, neither lipid affinity nor proteinprotein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-xL 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. We report that pro-apoptotic C-terminal cleavage fragments of Bcl-xL, but not their fulllength anti-apoptotic counterpart, permeabilize both mitochondria and pure lipid bilayer membranes to cytochrome c
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