Cytoprotection by Bcl-2 requires the pore-forming alpha5 and alpha6 helices.
We explored whether the putative channel-forming fifth and sixth alpha-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 alpha5-alpha6 were either deleted or swapped with each other, the Bcl-2Deltaalpha5alpha6 deletion mutant and Bcl-2-Bax(alpha5alpha6) 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 alpha5-alpha6 region of Bcl-2 is essential for its cytoprotective function. Additional experiments indicated that, although alpha5-alpha6 is necessary, it is also insufficient for the anti-apoptotic activity of Bcl-2. In contrast, deletion or substitution of alpha5-alpha6 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. BaxDeltaalpha5alpha6 and Bax-Bcl-2(alpha5alpha6) 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 alpha5-alpha6-dependent channels in membranes and to dimerize with and antagonize anti-apoptotic proteins such as Bcl-2.
Matsuyama
S
S
Burnham Institute, Program on Apoptosis & Cell Death Research, La Jolla, California 92037, USA.
Schendel
S L
SL
Xie
Z
Z
Reed
J C
JC
D017209:Apoptosis; D019281:Dimerization; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D007473:Ion Channels; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D019311:Staurosporine