Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization

Abstract

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2–associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Contribution of Yeast Studies to the Understanding of BCL-2 Family Intracellular Trafficking

“…This is an important observation showing that the overall capacity of BAX to permeabilize the OMM not only depends on its intrinsic ability to form pores, but also on its cellular content/stability. This is in line with a more recent study made in MEF [ 83 ].…”

“… Schematic representation of the link between BAX phosphorylation and Translocation. Phosphorylated BAX (by AKT and other survival kinases) is maintained away from mitochondria while non-phosphorylated BAX is in the OMM, both in yeast and mammalian cells [ 80 , 83 ]. The interaction of BAX with BCL-xL stimulate both BAX translocation [ 15 ] and BAX retrotranslocation [ 13 , 15 ].…”

Understanding and drugging the Bcl-2 transmembrane interactome for tumor treatment

“…The most promiscuous distribution is presented by Mcl-1 that localizes at the mitochondria (it appears into the MOM, the mitochondrial inner membrane and the mitochondrial matrix), the nucleus, the cytosol, and the ER, being accumulated into the MOM after apoptosis induction [65][66][67][68][69] . The proapoptotic protein Bax is mostly cytosolic in healthy conditions and translocates to the MOM upon apoptotic stimuli 43,70,71 , while Bak is constitutively inserted into the MOM and to a lesser extent into the ER membrane 72,73 . The third pore-forming member Bok targets the MOM, the nucleus, and the membranes of the ER and the Golgi apparatus [74][75][76] .…”

“…As mentioned before, almost all the multidomain Bcl-2 proteins and some BH3-only members target and insert into the intracellular membranes by their tail-anchor sequence or TMD. 70 . In addition to these observations, Bax G179E mutation also affects Bax proapoptotic activity.…”

“…This process is also reduced in the Bax T182I mutant promoting a predominantly mitochondrial localization. The T182 mutation makes Bax less efficient in oligomer formation and liposome membrane permeabilization 70 .…”