The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs

Abstract

Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was not spontaneously inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 promoted Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in H1 leading to Bax precipitation through hydrophobic interactions. The same motif was recognized by Tom22, triggering conformational changes leading to the extrusion and the ensuing membrane insertion of the C-terminal hydrophobic H9. Tom22 was also required for Bax-membrane insertion after Bax was activated either by BH3-activators or by its release from Bcl-xL by WEHI-539. The effect of Tom22 was impaired by D154Y substitution in Bax-H7 and T174P substitution in Bax-H9, that are found in several tumors. Conversely, a R9E substitution promoted the spontaneous insertion of Bax in nanodiscs, in the absence of Tom22. Both Tom22-activated Bax and BaxR 9E alone permeabilized liposomes to dextran-10kDa and formed [~]5nm-diameter pores in nanodiscs. The concerted regulation of Bax membrane insertion by Tom22 and BH3-activators is discussed.