Bax retrotranslocation potentiates Bcl-x'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, 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 strikingly revealed that the activity of Bcl-x 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 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 is necessary to strictly separate conditions of MOMP competency and resistance. Our results therefore identify Bax retrotranslocation by Bcl-x as an indispensable component of the molecular switch by which Bcl-2 family members govern cellular death decisions.