Bax Forms a Membrane Surface Protein-Lipid Complex as it Initiates Apoptosis

Abstract

Cellular clearance by apoptosis is essential in life. In its intrinsic (mitochondrial) pathway apoptotic members of the Bcl-2 (B cell CLL/lymphoma-2) protein family, such as Bax (Bcl-2-associated X) protein, perforate the mitochondrial outer membrane (MOM), which causes release of apoptotic factors and final cell death. How those apoptotic proteins mechanistically exert their action at the membrane level still however remains elusive. Upon internal stress signals Bax is massively recruited to the MOM, where it oligomerizes and partially penetrates into the membrane. Using neutron reflectometry (NR) and attenuated total reflection Fourier-Transform infrared spectroscopy (ATR-FTIR) spectroscopy we unraveled key molecular steps of this membrane-affiliation process of Bax on a spatial and temporal scale. By titrating intact human Bax to MOM-like bilayers containing cardiolipin, essential for protein recruitment, we could identify different functional phases. Initially, there is a fast adsorption event to the membrane surface with high affinity. Thereafter, a kinetically slower (minutes to hours) event occurs with Bax penetration, thereby triggering a major reorganizing of the mitochondrial bilayer. Finally, a membrane-Bax complex is generated, with a minor Bax population remaining membrane-inserted, while the main population is relocated to the membrane surface upon lipid redistribution into a complex with Bax; a process enabling membrane perforation. We propose a comprehensive molecular model of mitochondrial membrane penetration by formation of complex Bax/lipid clusters; a concept which provides a new foundation to understand the cell-killing activity of Bax and its apoptotic relatives in human cells.