BACKGROUND: Bcl-2 family proteins are key regulators of mitochondrial integrity 
and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member 
localizes as monomers in the cytosol of healthy cells and accumulates as 
oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain 
regulates interactions within the family, but regions other than BH3 are also 
critical for Bax function. Thus, the N-terminus has been variously implicated in 
targeting to mitochondria, interactions with BH3-only proteins as well as 
conformational changes linked to Bax activation. The transmembrane (TM) domains 
(alpha5-alpha6 helices in the core and alpha9 helix in the C-terminus) in Bax 
are implicated in localization to mitochondria and triggering cytotoxicity. Here 
we have investigated N-terminus modulation of TM function in the context of 
regulation by the anti-apoptotic protein Bcl-xL.
RESULTS: Deletion of 29 amino acids in the Bax N-terminus (Bax 30-192) caused 
constitutive accumulation at mitochondria and triggered high levels of 
cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30-105) 
abrogated mitochondrial localization but resulted in Bcl-xL regulated activation 
of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the 
alpha5-alpha6 helices/TMI domain (Bax 30-146) phenocopied Bax 30-192 as it 
restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not 
dependent on endogenous Bax-Bak. Inhibition of function and localization by 
Bcl-xL was restored in Bax 1-146, which included the TM1 domain. Regardless of 
regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated 
Bcl-xLand converged on caspase-9 dependent apoptosis consistent with 
mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence 
alignments of Bax and Bcl-xL indicated a sequence similarity between the 
alpha5-alpha6 helices of Bax and Bcl-xL. Alanine substitutions of three residues 
(T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the 
serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct 
regulatory mechanisms.
CONCLUSION: Collectively, the analysis of Bax deletion constructs indicates that 
the N-terminus drives conformational changes facilitating inhibition of 
cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as 
'structural antagonists' for BH3-Bcl-xL interactions, with this function being 
regulated by the N-terminus in the intact protein.