Bax retrotranslocation potentiates Bcl-x(L)’s antiapoptotic activity and is essential for switch-like transitions between MOMP competency and resistance

The rapid, typically all-or-none process of mitochondrial outer membrane permeabilization (MOMP) constitutes a primary cell death decision that is controlled by the Bcl-2 family interactome. However, how strict all-or-none MOMP decisions are governed by and emanate from the dynamic interplay of pro- and antiapoptotic Bcl-2 family members remains incompletely understood. In particular, it is unclear to which extent the shuttling of Bcl-2 family species between lipid and aqueous phases contributes to regulating MOMP sensitivity. Here, we studied the interplay of tBid, Bax, and Bcl-x(L), using a combined approach of deterministic mathematical modeling and retrospective as well as prospective experimental testing of model predictions. Systems modeling of the tBid–Bax interplay and their fluxes between cytosol and mitochondrial membranes reproduced experimental data on tBid-triggered Bax activation and oligomerization highly accurately. Extending these studies to analyze the cell-protective role of Bcl-x(L) strikingly revealed that the activity of Bcl-x(L) to retrotranslocate activated Bax from membranes back into the cytosol is essential to reproduce or correctly predict experimental outcomes. These included the potency of Bcl-x(L) in suppressing Bax oligomerization, its role in limiting Bax membrane recruitment, the resistance threshold to low concentrations of MOMP triggers as well as a response potentiaton arising from combinations of tBid and sensitizer BH3-only peptides. Importantly, retrotranslocation activity of Bcl-x(L) is necessary to strictly separate conditions of MOMP competency and resistance. Our results therefore identify Bax retrotranslocation by Bcl-x(L) as an indispensable component of the molecular switch by which Bcl-2 family members govern cellular death decisions.