Figure 5. BH1 and BH3 domains are required for the homo-oligomerization and proapoptotic activity of BAK.
(A) BH1 and BH3 domain mutants of BAK fail to undergo homo-oligomerization in response to tBID. Mitochondria isolated from DKO MEFs reconstituted with wt or mutant BAK were treated with recombinant tBID (1 ng/μl) and solubilized in 2 % CHAPS buffer. Protein lysates (200 μg) were subjected to Superdex 200 (HR 10/30) gel filtration chromatography and fractions were analyzed by anti-BAK immunoblots.
(B) BH1 and BH3 domain mutants of BAK fail to form homo-dimers. Radiolabeled IVTT N-terminal HA3-tagged wt or mutant BAK plus non-tagged counterparts were immunoprecipitated with anti-HA antibody. Immunoprecipitates and pre-IP input were analyzed by Nu-PAGE and autoradiography. Asterisk denotes degradation products.
(C) BH1 and BH3 domain mutants of BAK fail to trigger apoptosis. DKO MEFs reconstituted with wt or mutant BAK were treated with staurosporine (18 hr), etoposide (18 hr), tunicamycin (30 hr) or thapsigargin (30 hr) to induce apoptosis. Cell death was quantified by Annexin-V. Data shown are mean ± s.d. from three independent experiments.
(D) BH1 and BH3 domain mutants of BAK are localized at the mitochondria. Fluorescence microscopy of DKO MEFs stably reconstituted with YFP-tagged wt or mutant BAK followed by retroviral transduction of DsRed-Mito.
(E) BH1 domain is required for BAK to interact with tBID. Radiolabeled IVTT BAK wt, BAK BH3 mt (L75E) or BH1 mt (W122A/G123E/R124A) incubated with tBID-HA were immunoprecipitated with anti-HA antibody. Immunoprecipitates and pre-IP input were analyzed by Nu-PAGE and autoradiography.
(F) BAK constitutively exposes its N-terminal α1 helix. Mitochondria isolated from wt MEFs, mock treated or treated with tBID, were lysed in 1% CHAPS and then immunoprecipitated with the α1 helix specific anti-BAK antibody (NT). Immunoprecipitates and pre-IP input were analyzed by anti-BAK (G23) immunoblots. Mitochondria isolated from DKO MEFs serve as a negative control.
(G) BAK constitutively exposes its N-terminal α1 helix. 4T1 or N18 cells before or after staurosporine treatment were lysed in 1% CHAPS and then immunoprecipitated with the α1 helix specific anti-BAK antibody (NT). Immunoprecipitates and pre-IP input were analyzed by anti-BAK (G23) immunoblots.