Reconstitution of Proapoptotic BAK Function in Liposomes Reveals a Dual Role for Mitochondrial Lipids in the BAK-driven Membrane Permeabilization Process

Abstract

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.

The rheostat in the membrane: BCL-2 family proteins and apoptosis

“…Several reports have argued that curvature stress is the ultimate mechanism promoting the formation of protein-conducting membrane pores, and quite possibly the process can involve a cooperation between protein and lipid components to sculpt the membrane. 94,[96][97][98][99][100] Indeed, in cryo-EM images, Bax-induced pores in liposomes 92 and MOM vesicles (Kuwana and Newmeyer, personal This strongly suggests a pore formation or enlargement mechanism involving curvature stress. An alternative model was proposed by Martinez et al 101 after observing a BAX-dependent stepwise opening of highconductance membrane channels.…”

NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy

“…15 Externalization of CL to the mitochondrial surface is also part of other damage-related signaling pathways, including early pro-apoptotic signaling [16][17][18] and inflammatory reactions in intracellular compartments and extracellular environments. 19 By providing a scaffold at the mitochondrial surface for recruiting and functionally altering different apoptotic proteins, including caspase 8, tBid, Bak, and Bax, [20][21][22] CL participates in launching the release of cytochrome c, [23][24][25][26][27][28] although this step may not be required for all forms of apoptosis. 29 In contrast to the CL that triggers mitophagy through recognition via LC3, the CL externalized during apoptosis has mostly peroxidized acyl chains, due to the CL oxidase function of cytochrome c, 30 and this oxidation step seems to be important for execution of the apoptotic program.…”

Direct activation of full-length proapoptotic BAK

“…Instead, truncated forms of BAK have been generated and used in a series of studies that documented (i) dimeric structures of BAK truncates 23-185 and 16-186 (13, 14), (ii) binding of select BH3 domains to the exposed canonical pocket of BAKΔC (29) and N-and C-terminal calpain-cleaved BAK (cBAK) (14), (iii) autoactive and ligand-stimulated pore-forming functionality of an artificially membrane-tethered form of BAKΔC (30), and (iv) BH3 ligand-induced activation of BAK truncates in liposomal-and mitochondrial-release assays (29,31,32). The application of select BH3 peptides to native BAK-containing mouse liver mitochondria (33) and of in vitro transcribed/translated and tagged BH3-only proteins to mitochondria isolated from wild-type and mutant BAK-reconstituted Bax −/− Bak −/− mouse embryonic fibroblasts also supports a direct activation mechanism for BAK-mediated mitochondrial apoptosis (34,35).…”