Figure EV5. Related to Fig 4. Effect of membrane‐binding defective mutations on the Bax core dimerization.

• A
  A ribbon diagram of the NMR structure of bicelle‐bound Bax (α2–α5) dimer. One protomer is colored with the helices labeled. The C_α carbons of the residue pairs that were replaced by Cys pairs for disulfide crosslinking are shown as spheres linked by dashed lines. The R89E, A117D, and S118D mutations are represented by sticks.
• B–E
  [³⁵S]Met‐labeled Bax proteins with two Cys at position 59 and 79 (B, D) or one Cys at 69 (C, E) and an additional mutation, if indicated, to disrupt the α2–α5 core dimer interaction with membranes were synthesized, activated by Bax BH3 peptide, and targeted to the mitochondria lacking endogenous Bax and Bak proteins. The mitochondria‐bound proteins were oxidized by CuPhe for 30 min to induce disulfide crosslinking of the two protomers via the Cys pair(s) in the core dimer interface as shown in (A). The radioactive crosslinked Bax dimer was then separated from the monomer by non‐reducing SDS–PAGE and detected by phosphor‐imaging. The representative data from two to four independent experiments are shown. The relative dimer:monomer ratio shown at the bottom of the phosphor‐images was determined by the intensities of the dimer and monomer bands of the Cys mutant with the additional mutation, normalized to that of the corresponding Cys mutant without the additional mutation. The mitochondrial association efficiency of each Bax mutant (E) was determined by the summed intensity of the monomer and dimer bands in the mitochondrial fraction that was divided and treated with CuPhe for 0 or 30 min (I_M), the intensity of the monomer band in the corresponding soluble fraction that was divided by 0.015 to adjust the inequivalent loading between the mitochondrial (1 equivalent (eq)) and soluble (0.015 eq) fractions (I_S), and the following equation, ${\text{E = I}}_{\mathrm{M}}/({\mathrm{I}}_{\mathrm{M}}+{\mathrm{I}}_{\mathrm{S}})$. The relative mitochondrial association efficiency of each Bax Cys mutant with the additional mutation (E_R) to that of the Bax Cys mutant without the additional mutation was obtained by dividing the E of the former with the E of the latter and shown at the bottom of the phosphor‐images.

Source data are available online for this figure.