Abstract
Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.
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Abbreviations
- BH3:
-
Bcl-2 homology 3
- BMOE:
-
1,6-bis-maleimidoethane
- C terminus:
-
carboxy terminus
- CuPhe:
-
copper(II)(1,10-phenanthroline)3
- FRET:
-
Förster resonance energy transfer
- GFP:
-
green fluorescent protein
- IASD:
-
4-acetamido-4′-((iodoacetyl)amino)stilbene-2,2′-disulfonic acid
- IEF:
-
isoelectric focusing
- IRES:
-
internal ribosome entry site
- MEFs:
-
mouse embryonic fibroblasts
- MOA:
-
monoamine oxidase A
- MOM:
-
mitochondrial outer membrane
- SDS-PAGE:
-
SDS-polyacrylamide electrophoresis
- S/N:
-
supernatant
- tBid:
-
truncated Bid
- wt:
-
wild type
- TMD:
-
transmembrane domain.
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Acknowledgements
We thank Peter Colman and Rachel Uren for critical comments on the manuscript. We also thank Matthew Call and Melissa Call for advice on generating the C terminus swap mutants, Peter Czabotar for useful discussions, Ray Bartolo and Stephanie Fennell for technical support. GD and RMK acknowledge ARC Future Fellowships. Our work is supported by NHMRC grants (637337 and 1016701), and the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS.
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Iyer, S., Bell, F., Westphal, D. et al. Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains. Cell Death Differ 22, 1665–1675 (2015). https://doi.org/10.1038/cdd.2015.15
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DOI: https://doi.org/10.1038/cdd.2015.15
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