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Protocol

Investigating the Oligomerization of Bak and Bax during Apoptosis by Cysteine Linkage Grant Dewson1 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

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)(1,10phenanthroline)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.

MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Cross-linking buffer (for chemical cross-linking) Cysteine linkage reagent of interest Chemical cross-linker (bismaleimidoethane [BMOE] or bismaleimidohexane [BMH], prepared at a concentration of 10 mM on the day of the experiment) (Pierce) Chemical cross-linkers provide an alternative to disulfide-bonding for monitoring protein oligomerization by cysteine linkage. These reagents come in many different “flavors,” with different linker arm lengths, cleav-

1

Corresponding: [email protected]

© 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086470

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G. Dewson ability and solubility. An excellent resource for selecting the appropriate cross-linking reagent is Piercenet .com. BMOE and BMH have 8 and a 12 Å linkers, respectively, and therefore will cross-link cysteines within different distance constraints. Both reagents are cell-permeable and thus can be applied directly to cells in culture. As they are uncleavable, samples are processed as normal for reducing SDS–PAGE.

Copper (II)(1,10-phenanthroline)3 (CuPhe) (10 mM) Disulfide linkage using CuPhe is both rapid and efficient; because it is effectively a “zero-length” crosslinker, it can provide detailed information about the conformation or interfaces of a protein. However, due to the relative positions of the endogenous cysteines in Bak and Bax, oxidation is an informative approach only for the former, while longer-length cross-linkers are required for analysis of Bax.

Membrane fraction pellet, prepared as described in Steps 1–6 of Protocol: Investigating Bax Subcellular Localization and Membrane Integration (Dewson 2015) As Bak and Bax oligomerize at the mitochondrial outer membrane, the cytosolic fraction (supernatant at Step 6) can be discarded. Note that permeabilization and separation of the membrane fraction from the cytosol are not absolutely necessary, as CuPhe, BMH, and BMOE are all cell-permeable; however, these steps improve detection of disulfide-linked Bak oligomers. Alternatively, whole cells may be used.

Permeabilization buffer (for disulfide linkage) Protease inhibitor cocktail tablets, without EDTA (cOmplete; Roche) (1 tablet/50 mL) SDS–PAGE and immunoblotting reagents, including primary antibodies for Bak and/or Bax SDS–PAGE sample buffer (nonreducing) (2×) (for disulfide linkage) Supplement nonreducing sample buffer with 100 mM EDTA before use (Step 3).

SDS–PAGE sample buffer (reducing) (2×) (for chemical cross-linking) Equipment

Heat block at 95oC SDS–PAGE apparatus Western transfer apparatus METHOD In this protocol, cells are treated to induce cysteine linkage using one of two methods: The induction of disulfide linkage by oxidation for investigation of Bak (see Steps 1–3) or the use of chemical cross-linkers for investigation of both Bak and Bax (see Steps 4–6).

Inducing Disulphide Linkage

1. Resuspend the membrane fraction pellet in permeabilization buffer (with protease inhibitors but without digitonin) at 1 × 107 cells per mL. The membrane fraction may be difficult to resuspend in permeabilization buffer at this stage. It is helpful to pipette up and down several times with a P200 pipette until no clumps are visible.

2. Add 10 mM CuPhe to each sample to a final concentration of 1 mM. Incubate for 30 min on ice. 3. Add an equal volume of 2× nonreducing SDS–PAGE sample buffer supplemented with 100 mM EDTA (to chelate the copper). Proceed to Step 7. Do not add a reducing agent (DTT or β-mercaptoethanol) at any stage, as it will inhibit/disrupt disulfide linkage.

Cross-Linking with a Chemical Cross-Linker

4. Resuspend the membrane fraction pellet in cross-linking buffer at 1 × 107 cells per mL. 5. Add 10 mM BMH or BMOE to a final concentration of 0.5 mM. Incubate for 30 min in the dark at room temperature. When adding BMH or BMOE in DMSO, the final DMSO concentration must be

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 ...
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