Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID

BID is a BH3 domain-only member of the Bcl-2 family that acts as an apoptot ic agonist in programmed cell death. After cleavage by caspase-8, the N-ter minal of BID (N-BID) stays in the cytosol while the C-terminal of BID (C-BI D) translocates to mitochondria, leading to cytochrome c release in vivo an d in vitro. We have previously reported that BID or truncated BID (tBID) ca n induce the release of entrapped trypsin and cytochrome c from large unila mellar vesicles (LUVs). Further studies have been performed and are present ed here: the results demonstrate that C-BID, like BID and tBID, induces ves icle leakage, whereas N-BID or the BID mutants BID (D59A) and BID (G94E) fa il to have any significant effects. The affinity of the above-mentioned pro teins for soybean phospholipid LUVs (SLUVs) decreased in an order similar t o their leakage-inducing capability: tBID > BID > BID (D59A), while N-BID a nd BID (G94E) were unable to bind to the vesicles at all. BID-induced leaka ge was dependent on the lipid composition of vesicles. Acidic phospholipid (e.g. phosphatidic acid or phosphatidylglycerol) was necessary for BID-indu ced leakage while the presence of phosphatidylethanolamine or cholesterol r educed the leakage. It was also found C-BID is better able to penetrate the soybean phospholipid monolayer than BID or tBID. A further finding was tha t tBID, but not full-length BID, could stimulate the aggregation of SLUVs. Finally, Bcl-x(L). an apoptotic antagonist in programmed cell death, can pr event the aggregation of LUVs induced by tBID, but not the release of entra pped trypsin. It is postulated that two separate domains of tBID are respon sible for inducing leakage and aggregation of phospholipid vesicles.