Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition

Pro-survival factor Bcl-XL can antagonize the pro-apoptotic functions of Bax and Bad via two distinct mechanisms. It can block Bax-mediated cell death by preventing Bax translocation from the cytosol to mitochondria. On the other hand, Bcl-XL can neutralize Bad by sequestering it to mitochondria. In order to map the domains of Bcl-XL involved in inhibiting Bax and Bad, we have carried out mutational analyses of this protein. This was done by deleting the key domains of Bcl-XL, including its BH1-4 domains, the flexible loop, the C-terminal hydrophobic domain, and segments of the α5-α6 hairpin. The resulting Bcl-XL mutant constructs were then co-transfected with either GFP-Bax or GFP-Bad. We found that the BH1-4 domains and the C-terminal segment of Bcl-XL were essential for blocking Bax localization to mitochondria. On the other hand, only its BH1 and BH3 domains and the C-terminal hydrophobic segment were necessary for sequestering Bad to mitochondria. In addition, by immunoprecipitation analyses, we found that these deletions differentially affected the ability of the Bcl-XL mutant proteins to bind Bax and Bad. Finally, cell viability assays indicated that the BH1-4 domains of Bcl-XL were the primary domains required for inhibiting staurosporine-induced apoptosis, suggesting that distinct domains of Bcl-XL are involved in antagonizing Bax and Bad and in apoptosis inhibition.