Bak and Bax are the critical effectors of intrinsic apoptotic cell death responsible for permeabilising the mitochondrial outer membrane during apoptosis. The ‘fine tuning’ of these proteins by other members of the Bcl-2 family acts as a molecular switch that commits the cell to death. We have shown that Bak and Bax reside in large multisubunit complexes in mitochondria that comprise non-Bcl-2 proteins including VDAC2¹ . The components of these complexes represent novel regulators of Bak and Bax function. To identify these putative regulators we established an enzymatic tagging approach to interrogate the interacting partners of the death effectors Bak and Bax.

Proximity-induced biotinylation utilises a mutant version of the bifunctional ligase/repressor (BirA) from E. coli that is able to non-specifically conjugate proximal proteins within 10-20 nm with a biotin moiety² . Biotinylation modifications are rare and extremely stable in mammalian cells, making them ideal for high purity enrichment for downstream analysis. We then analysed these samples by mass spectrometry to identify putative proximal interacting partners of Bak and Bax in the presence or absence of an apoptotic stimulus.

Our studies have revealed a number of candidate proteins that are selectively interacting with Bak in a healthy setting and are subsequently lost after induction of an apoptotic stimulus. These represent potential novel regulators of Bak and Bax. We intend to further validate these proteins with a series of biochemical assays including immunoprecipitation and immunocytochemistry to validate that they do indeed represent functional regulators of Bak and Bax.

This proximity-dependent enzymatic tagging approach is a robust, sensitive system that can be adapted to interrogate a host of protein-protein interactions, including those that are transient and labile, in a variety of biological settings.