Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x_L. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.Bax and Bak are the key effectors of the intrinsic apoptotic pathway initiated in response to diverse stimuli including anoikis, DNA damage and growth factor withdrawal.¹ Both proteins are normally dormant in healthy cells, but upon reception of an apoptotic stimulus, they undergo conformation change that allows their self-association to form pores in the mitochondrial outer membrane (MOM).^{2, 3, 4, 5, 6, 7} The consequence of disruption of the MOM is twofold; it impairs the ability of mitochondria to generate ATP by oxidative phosphorylation and it allows the release of intermembrane proteins including cytochrome c that agonizes caspases that dismantle the cell.Bak and Bax share significant structural homology in their inactive states and have conserved mechanism of conformation change and oligomerization.^{3, 8, 9, 10} Further, genetic studies reveal that Bak and Bax perform at least partially overlapping function, with deficiency in both necessary to perturb apoptosis during embryonic development and in response to toxic insult.^{1, 11} However, whether Bak and Bax are regulated similarly is unclear. Whereas Bak is constitutively anchored in the MOM via its hydrophobic C-terminal transmembrane domain, Bax is predominantly cytosolic in the majority of non-apoptotic cells.¹² Recent evidence indicates that Bax is in a dynamic equilibrium between cytosol and mitochondria and is constantly trafficked away from the MOM in non-apoptotic cells.^{13, 14} In response to apoptotic stress this ‘retrotranslocation'' is disrupted causing Bax to accumulate at mitochondria; a hallmark of most apoptotic cells. The mechanism governing the dynamic distribution of Bax in healthy and apoptotic cells is unclear with interactions with pro-survival proteins debated.^{13, 14}Voltage-dependent anion channels (VDACs) are the major channels responsible for ion passage across the MOM. Studies have also implicated an additional role for the VDACs in the regulation of Bak or Bax apoptotic function or potentially even constituting a component of the Bak/Bax apoptotic pore.^{15, 16, 17, 18} However, these studies have provided contrasting findings relating to whether VDACs might positively or negatively regulate Bak/Bax apoptotic function.We used blue native-PAGE (BN-PAGE) to investigate how Bax oligomerizes in the MOM during apoptosis. We observed that VDAC2 is a determinant of the constitutive association of both Bax and Bak with the MOM. The defect in Bax mitochondrial localization can be bypassed by Bak-dependent recruitment during apoptosis. Thus, our data suggest that mitochondrial localization of Bax occurs via distinct mechanisms in healthy and apoptotic cells and that either VDAC2 or Bak is required for the efficient translocation of Bax and hence for the oligomerization at the MOM and Bax apoptotic function.