FIG. 4.

Specificity of the LTP-like activity displayed by recombinant Bid. (A) Donor and acceptor liposomes were mixed first for over 10 min under the conditions of the experiment shown in Fig. 2B to reach equilibrium in the spontaneous lipid transfer. A total of 1 nM (25 ng/ml) recombinant human Bid was then added as indicated, and the fluorescence changes were recorded for about 10 min. The top trace was obtained in the absence of other additions, while the bottom trace was obtained in the presence of 25 μg of BSA per ml which strongly inhibits lipid transfer (5, 33). Subsequently, 50 μg of freeze-thawed pig heart mitochondria per ml (equivalent to approximately 20 μg of membrane lipids per ml) was added to further stimulate lipid transfer. The final concentration of the lipid probe was 50 nM. (B) The increase in fluorescence of the PA analog probe was measured at 1 h after the addition of pig heart mitochondria in experiments similar to those shown in panel A. Subsequently, the reaction mixtures were centrifuged at 10,000 × g for 15 min to separate the mitochondria, and the pellets were resuspended in assay buffer containing 0.5% (vol/vol) Triton X-100 and then diluted in the cuvette. The fluorescence recovered in the pellets was measured as described in the legend to Fig. 2. The bottom spectrum was obtained with mitochondria alone (without the lipid probe), the intermediate spectrum was obtained with acceptor and donor liposomes plus Bid, and the top spectrum was obtained with donor and acceptor liposomes plus Bid and mitochondria. (C) Isolated native Bid (10 nM) (filled symbols) or recombinant Bak (10 nM) (open symbols) was incubated with donor liposomes, and lipid transfer was monitored by time-resolved spectra after addition of 3.5 μg of acceptor liposomes per ml as in Fig. 2B. The time course of the intensity in the emission maximum (at 515 nm; F515) is shown. The spontaneous transfer to the acceptor liposomes was essentially equivalent to that measured in the presence of Bak and is not shown, for the sake of clarity. Similar results were obtained using pig heart mitochondria as acceptors (not shown). (D) Caspase 8-cleaved Bid (tBid) and its parent full-length preparation of recombinant mouse Bid were added to donor liposomes (obtained by sonication as shown in Fig. 3) diluted to a final concentration of 30 nM BODIPY FL C5-HPA. After approximately 3 min, 2.5 μg of LUV acceptor liposomes per ml was added, and the initial rate of lipid transfer was recorded as shown in Fig. 3A. Similar differences in the rate of lipid transfer of tBid and full-length Bid were seen in a wide range of protein concentrations up to 200 nM (not shown). a.u., arbitrary units.