Background: During apoptosis Bax/Bak release differently sized proteins out of the mitochondria. Results: The size of Bax/Bak pores depends on protein concentration. Conclusion: Bax/Bak form stable toroidal pores tunable in size. Significance: Pore size-tuning constitutes a new level for the regulation of Bax/Bak activity.

BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAKdriven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAK⌬C) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAK⌬C membrane insertion, oligomerization, and pore formation. Furthermore, we demonstrate that tBID-activated BAK⌬C permeabilizes the membrane by forming structurally dynamic pores rather than a large proteinaceous channel of fixed size. We also identified two distinct roles played by mitochondrial lipids along the molecular pathway of BAK⌬C-induced membrane permeabilization. First, using several independent approaches, we showed that cardiolipin directly interacts with BAK⌬C, leading to a localized structural rearrangement in the protein that "primes" BAK⌬C for interaction with tBID. Second, we provide evidence that selected curvatureinducing lipids present in mitochondrial membranes specifically modulate the energetic expenditure required to create the BAK⌬C pore. Collectively, our results support the notion that BAK functions as a direct effector of MOMP akin to BAX and also adds significantly to the growing evidence indicating that mitochondrial membrane lipids are actively implicated in BCL-2 protein family function.

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOMlike liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.