Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine Linkage.
Following conformation change, Bak and Bax self-associate to form the putative apoptotic pore in the mitochondrial outer membrane. The nature of this pore and whether it is purely proteinaceous or lipidic are still unresolved. Induction of disulfide linkage with oxidants such as copper (II)(1,10-phenanthroline)3 (CuPhe) and chemical cross-linking with cell-permeable homobifunctional maleimide reagents are convenient ways to investigate Bak and Bax oligomerization in cells or isolated mitochondria. A limitation of these methods is they are based on the linkage of cysteines, and their success is reliant on the positions of the endogenous cysteines in Bak and Bax. Consequently, the protocols are more efficient and informative for human Bak than that for its murine counterpart. An additional benefit when investigating human Bak is that cysteine-based linkage assays provide information on the conformation change that precedes Bak oligomerization: Endogenous cysteines in the inactive form are in close proximity, and intramolecular linkage after treatment causes inactive Bak to migrate faster during SDS-PAGE. This intramolecular linkage is lost on activation, as the cysteines are distanced by conformation change. During apoptosis, Bak oligomerization induces the proximity of cysteines that favor intermolecular linkage. Trapped Bak oligomers can be detected with nonreducing (following oxidation with CuPhe) or reducing (following chemical cross-linking with homobifunctional maleimide reagents) SDS-PAGE and immunoblotting, as described here.
Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia;Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
Grant
G
Dewson
D000818:Animals; D017209:Apoptosis; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D055503:Protein Multimerization; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D004220:Disulfides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D003545:Cysteine