Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.
Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
Mitochondria are the key energy-producing structures inside cells, but are also crucial players in a common form of programmed cell death, apoptosis. A critical event in mitochondrion-driven apoptosis involves the formation of large pores in the mitochondrial outer membrane (MOM). These pores cause long-term damage to mitochondria and also allow mitochondrial proteins to escape and accelerate cell death. Previous studies have revealed that the protein Bax when activated can form pores in protein-free membranes and that it, along with Bak, is involved in the formation of mitochondrial pores, but the process remains unclear. We now show, however, that in naturally derived MOMs, Bax is assisted by another resident MOM protein, which we term the “catalyst,” and whose identity is still unknown. The mechanism involves two distinct stages. First, activated Bax activates the catalyst protein, causing multiple catalyst molecules to assemble into a larger structure (a complex). In the second stage, this catalyst complex in turn facilitates Bax-driven pore formation. Our data also reveal some unexpected details of the pore formation process; in particular, it appears that catalyst activation involves a physical change in the molecular arrangement of the membrane. Furthermore, contrary to what was previously assumed, pore formation does not require Bax molecules themselves to assemble together into larger complexes.