Bak and Bax mediate the mitochondrial pathway of apoptosis by oligomerising to form an apoptotic pore in the mitochondrial outer membrane (MOM), although the mechanism by which oligomers disrupt the lipid bilayer is unknown. Bak and Bax are both targeted to the MOM by their C-terminal transmembrane domain (TMD), which inserts into the membrane. However, there is no information on the topology of the TMD in the apoptotic pore complex and whether a TMD:TMD interface might contribute to pore function.
In this study we employed a cysteine linkage approach to determine whether a TMD:TMD interface is present in Bak before and/or after MOMP. Cysteine residues were substituted at each position throughout the Bak TMD helix. Bak variants were then stably expressed in bak-/-bax-/- mouse embryonic fibroblasts and tested for membrane insertion and function. Cysteine labeling with a membrane impermeable alkylating reagent verified its transmembrane nature. Linkage of cysteines in mitochondrial fractions revealed an interface between the Bak TMDs after, but not before apoptotic signaling. Moreover, linkage at only certain TMD cysteines suggested limited rotational freedom within the membrane. Bax also showed TMD:TMD linkage at the MOM after apoptotic signaling. We also further examined the requirement of the native C-terminus of Bak for apoptotic function by tail anchor substitutions.
Thus, we have identified a novel TMD:TMD interface in the apoptotic pore complex as well as its relationship with other interfaces in Bak. Also the requirement of the C-terminus for apoptotic function was investigated.