Cytoprotection by Bcl-2 requires the pore-forming and helices

Abstract

We explored whether the putative channel-forming fifth and sixth α- helices of Bcl-2 and Bax account for Bcl-2-mediated cell survival and Bax- induced cell death in mammalian cells and in the yeast Saccharomyces cerevisiae. When α5-α6 were either deleted or swapped with each other, the Bcl-2Δα5α6 deletion mutant and Bcl-2-Bax(α5α6) chimeric protein failed to block apoptosis induced by either Bax or staurosporine in human cells and were unable to prevent Bax-induced cell death in yeast, implying that the α5-α6 region of Bcl-2 is essential for its cytoprotective function. Additional experiments indicated that, although α5-α6 is necessary, it is also insufficient for the anti-apoptotic activity of Bcl-2. In contrast, deletion or substitution of α5-α6 in Bax reduced but did not abrogate apoptosis induction in human cells, whereas it did completely nullify cytotoxic activity in yeast, implying that the pore-forming segments of Bax are critical for conferring a lethal phenotype in yeast but not necessarily in human cells. BaxΔα5α6 and Bax-Bcl-2(α5α6) also retained the ability to dimerize with Bcl-2. Bax therefore may have redundant mechanisms for inducing apoptosis in mammalian cells, based on its ability to form α5-α6- dependent channels in membranes and to dimerize with and antagonize anti- apoptotic proteins such as Bcl-2.