Abstract
During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.
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Abbreviations
- BH3:
-
Bcl-2 homology 3
- BN-PAGE:
-
blue native PAGE
- CuPhe:
-
copper(II)(1,10-phenanthroline)3
- DTT:
-
dithiothreitol
- GFP:
-
green fluorescent protein
- HA:
-
haemagglutinin
- hBak:
-
human Bak
- hBax:
-
human Bax
- IRES:
-
internal ribosome entry site
- LOF:
-
loss-of-function
- MEFs:
-
mouse embryonic fibroblasts
- MOM:
-
mitochondrial outer membrane
- tBid:
-
truncated Bid
- TNF-α:
-
tumour necrosis factor alpha
- wt:
-
wild-type
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Acknowledgements
We thank Peter Colman for comments on the manuscript, Michael Ryan and Michael Lazarou for advice on BN-PAGE, Stephanie Fennell and Laura Raiti for technical assistance, and David Huang and W Douglas Fairlie for reagents. The work was supported by grants from the National Health and Medical Research Council of Australia (no. 575559 and no. 637335), and the Association for International Cancer Research (no. 10–230), and operational infrastructure grants through the Australian Government IRISS and the Victorial State Government OIS.
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Dewson, G., Ma, S., Frederick, P. et al. Bax dimerizes via a symmetric BH3:groove interface during apoptosis. Cell Death Differ 19, 661–670 (2012). https://doi.org/10.1038/cdd.2011.138
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DOI: https://doi.org/10.1038/cdd.2011.138
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