The Bax carboxy-terminal hydrophobic helix does not determine organelle-specific targeting but is essential for maintaining Bax in an inactive state and for stable mitochondrial membrane insertion.

Here we address the function of the hydrophobic carboxy-terminal tail of the pro-apoptotic protein Bax. The tail is tucked into a hydrophobic pocket within the closed/inactive conformation of Bax. Apoptotic stimulation changes the Bax conformation, exposing a mitochondrial-targeting signal. We confirmed that the Bax tail alone can specifically target and anchor a passenger protein to the mitochondria. Surprisingly, we determined that the Bax tail does not play the primary targeting role in Bax mitochondrial translocation. Mutating the Bax tail to produce an ER-targeting signal had no effect on Bax mitochondrial targeting. Additionally, we demonstrated that the Bax tail has a negative regulatory effect on Bax activation. Mutations that disrupt the tail interactions with the hydrophobic pocket resulted in constitutive activation and mitochondrial targeting. Deletion of the Bax tail also resulted in an active conformation of Bax, however, mitochondrial targeting was abolished. Thus, the Bax tail is required for mitochondrial translocation. By generating a mutant-tail that cannot insert into membrane, we determined that insertion of the Bax tail is required for Bax mitochondrial targeting. Our data support a model whereby the Bax tail must be released from the pocket for activation of Bax, then functions as an anchor to stabilize Bax at the mitochondrial membrane after the initial addressing step.