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. 2010 Jul 6;285(37):28924–28937. doi: 10.1074/jbc.M110.135293

FIGURE 2.

FIGURE 2.

Permeabilization of membrane by membrane-targeted BAK and Bid. A, schematic representation of mouse BAK protein constructs. Soluble form of mouse BAK protein (sBAK, residues 16–184) was prepared with (sBAK-ΔC-His) or without a C-terminal hexahistidine tag as described under “Experimental Procedures.” sBAK/G124V-ΔC-His and sBAK/C154S-ΔC-His represent sBAK-ΔC-His, in which residues Gly-124 and Cys-154 are substituted to valine and serine, respectively. B, schematic representation of the liposome dye release assay. The C-terminally His6-tagged sBAK (sBAK-ΔC-His) is targeted to the membrane via the binding of the His6 tag to the Ni2+-NTA moiety on the surface of the LUVs containing 5% (w/w) DOGS-NTA-Ni and other lipids mimicking the mitochondrial outer membrane contact sites (Ni-Nta-MCS LUVs) (see “Experimental Procedures”) (39). tBid (p15Bid without His6 tag), His6 tagged p7/p15 Bid, or His6-tagged Bid BH3 also targets to the membrane and exposes its BH3 domain, promoting BAK to form oligomeric pores, the conduit for the encapsulated FITC-dextran (10 kDa) release into the medium, resulting in fluorescence dequenching. C, effect of the hexahistidine tag, tBid, metal chelator, and the G124V amino acid substitution mutation on the membrane permeabilization by soluble BAK (sBAK). In the presence or absence of 5 mm EDTA, tBid (5 nm) and/or soluble BAK (10 nm) with (sBAK-ΔC-His) or without the C-terminal His6 tag (sBAK) were added at the indicated times to the Ni-Nta-MCS LUVs (10 μg/ml, or 0.125 nm (32)) encapsulating FITC-dextran (10 kDa). D, effect of Bcl-XL, an antiapoptotic Bcl-2 family protein on the tBid-catalyzed membrane permeabilization by soluble BAK. In the presence or absence of wild type or mutant form (m8) of 200 nm Bcl-XL fusion protein (GST-Bcl-XL-ΔC-His (wt) or (m8)), tBid (5 nm) and sBAK-ΔC-His (10 nm) were added at the indicated times to the Ni-Nta-MCS LUVs (10 μg/ml) encapsulating FITC-dextran (10 kDa). Bcl-XL (m8) mutant protein contains G138E/R139L/I140N triple amino acid substitution mutations. E, effect of tBid, His6-tagged Bid BH3 peptide, and the N-terminally His6-tagged p7/p15 Bid on the membrane permeabilization by the membrane-targeted BAK. tBid, His-Bid BH3 peptide, or His-p7/p15 Bid (all at 50 nm) and/or sBAK-ΔC-His (2 nm) were added at the indicated times to the liposomes (10 μg/ml) encapsulating FITC-dextran (10 kDa). C–E, all the experiments were carried out at 37 °C with the Ni-Nta-MCS LUVs containing 5% (wt) DOGS-NTA-Ni.