The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.
- Bax inactivation
- calcium promotion of leakage
- hydrophobic binding site
- membrane translocation
Abbreviations used: ANTS, 8-aminonaphthalene-1,3,6-trisulphonic acid; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulphonic acid; DNS-PE, N-[5-(dimethylamino)naphthalene-1-sulphonyl]-l-α-phosphatidylethanolamine; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; DOPG, dioleoylphosphatidylglycerol; DPX, p-xylene-bis-pyridinium bromide; DTT, dithiothreitol; LUV, large unilamellar vesicle; tBid, caspase-8-cut Bid.
- The Biochemical Society, London ©2002