Comparison of BH3-dependent and BH3-independent membrane interactions of pro-apoptotic factor BAX

The pro-apoptotic factor BAX is a key member of the B cell lymphoma-2 family of apoptotic regulators. BAX functions by permeating the mitochondrial outer membrane, a process that begins with the targeting of soluble BAX to the membrane. Once associated, BAX refolds, inserts into the bilayer, and ultimately assembles into a multimeric pore of unknown structure. BAX targeting is initiated by an activation signal that can arise from two pathways: 1) a BH3-dependent one in which BAX is activated by one of the BH3-only effectors, such as tBid, or 2) a recently discovered BH3-independent pathway, where BAX activity is modulated by changes in lipid composition. In this study, we gain further insight into how these two pathways function and how their function is impacted by anti-apoptotic factor Bcl-xL. We use fluorescence spectroscopy to compare the BH3-dependent and BH3-independent interactions of BAX with model membranes of varying lipid compositions. We investigate membrane association using Förster resonance energy transfer between donor-labeled BAX and acceptor-labeled vesicles. We monitor membrane insertion by observing changes in the spectral properties of the environment-sensitive probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), which we selectively attached to a series of single-cysteine BAX mutants. Finally, we study membrane permeation through BAX-induced leakage of soluble markers loaded into vesicles. Our results show that BAX-induced permeabilization of zwitterionic vesicles is more efficient for the BH3-dependent pathway than the BH3-independent pathway; however, permeabilization of cardiolipin-containing vesicles is equally efficient for both the BH3-dependent and BH3-independent pathways. Interestingly, although anionic lipids are not necessary for the initial BH3-independent membrane association of BAX, they are critical for subsequent stages of membrane insertion and pore assembly. The spectroscopic response of NBD-labeled BAX is comparable for both interaction modes, indicating a similar structure for the final inserted state. We found that the Bcl-xL factor inhibits vesicle permeabilization by preventing BAX from interacting with the bilayer.