Bax Initiates the Assembly of a Multimeric Catalyst that Facilitates Pore Formation in Mitochondrial Outer Membranes

Abstract

In vertebrates, apoptotic cell death involves a canonical “intrinsic” apoptotic pathway that depends on mitochondrial outer membrane permeabilization (MOMP). MOMP is mediated by the pro-apoptotic Bcl-2 family proteins Bax and/or Bak. Previous studies have modeled Bax-dependent membrane pore formation in simple in vitro systems consisting of protein-free synthetic liposomes mixed with recombinant Bax and “direct activator” BH3-only proteins, e.g. cBid. However, it has been unclear whether liposome systems accurately reflect the physiological events in mitochondria. To determine whether Bax requires the assistance of other membrane proteins to form pores, we analyzed the kinetics of MOMP using isolated mitochondrial outer membranes incubated with recombinant Bax, along with cBid. Compared with liposomes, native membranes were much more sensitive to Bax and displayed more complex permeabilization kinetics. Heat-labile outer membrane 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 serves as catalyst for the second reaction, pore formation. The kinetics was affected in a reciprocal manner by [cBid] and [Bax], indicating that cBid-induced Bax activation is governed by kinetic mass action law, consistently with the “hit-and-run” mechanism. Strikingly, MOMP rate constants were linearly related to [Bax], implying lack of Bax cooperativity. Thus, contrary to popular assumption, pore formation kinetics does not depend on Bax oligomerization. Moreover, our data show that assembly of the catalyst complex depends on membrane fluidity and is blocked by chemical inhibitors of Drp1-dependent mitochondrial fission. However, Drp1 itself was undetectable in mitochondrial outer membranes. Thus, the data suggest that a noncanonical Drp1-like activity facilitates Bax-induced MOMP, possibly through a membrane-remodeling event.