Bax Enhances the Permeabilization of the Mitochondrial Outer Membrane Induced by Ceramide Channels: Implications on the Regulation of the Initiation of Apoptosis

Abstract

Background: Bax is a pivotal pro-apoptotic Bcl-2 family protein that localizes to the mitochondrial outer membrane (MOM) during apoptosis and causes MOM permeabilization to proteins (MOMP). Earlier studies have demonstrated that upon an apoptotic stimulus, ceramide levels often greatly increase in cell membranes, including in the MOM. Elevation of ceramide in the MOM is sufficient to cause MOMP without requiring Bcl-2 family proteins. Moreover ceramide induced MOMP is reversed/prevented by the anti-apoptotic protein, Bcl-xL. Methods: Using rat liver or yeast mitochondria, the MOMP was measured with a dynamic cytochrome c accessibility assay. Ceramide's channel-forming ability was also assessed using a defined system: planar phospholipid membranes. Only C16-ceramide was used. Results: We found that Bax induces MOMP by apparently enlarging ceramide channels. While ceramide forms channels independently of Bax, the permeabilization is enhanced by the addition of less than 5nM oligomeric Bax. As much as 50nM oligomeric Bax alone did not result in any significant MOMP. The Bax enhancement occurs with an apparent affinity that increases with an increase in ceramide-induced MOMP, indicating an underlying mechanism by which Bax enhances ceramide-induced MOMP. Bax also causes apparent ceramide channel enlargement in yeast mitochondria, which lack Bcl-2 family proteins, as well as in planar phospholipid membranes, which is a defined, protein free, system. By contrast, monomeric Bax has no effect on ceramide channels in the aforementioned systems. The Bax inhibitor, Bci2 [Bruno Antonsson], prevents Bax mediated channel enlargement but does not affect permeabilization induced by ceramide alone. Conclusions: Both pro- and anti-apoptotic proteins regulate ceramide channels, consistent with ceramide channels being the pathway by which proteins are released by mitochondria early in apoptosis. (Supported by NSF grant: MCB-0641208)