Figure 3.
Bcl-xL association with inner mitochondrial membrane components. (A) Summary of yeast two-hybrid interactions between BCL-2 family proteins and the ATP synthase β subunit, including wild-type (WT) and Bcl-xL mutants mt1 (F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B) Yeast (BY4741) transformed with the indicated plasmids were heat ramp treated and presented as colony counts from four determinations in two independent experiments. Data are presented as the mean ± SEM. Student’s t test was used; *, P = 0.012 compared with control; **, P = 0.001. (C) Immunoblots for Bcl-xL (provided by L. Boise) or β subunit in total rat liver mitochondria (T), inner membrane vesicles (F1), and highly purified ATP synthasomes (F2 and F3) of increasing purity (Ko et al., 2003). The replicate blot in the middle was probed with Bcl-xL antibody preincubated with recombinant Bcl-xL (rBcl-xL) protein purified from Escherichia coli (lanes F1–F3 only). (D) Coomassie blue–stained preparative SDS gel of final purification step for Bcl-xL–binding partners from WEHI 7.1 cells. (E) Immunoblots for Bcl-xL and β subunit in size column chromatography fractions (F) and total lysates (T). Eluted size markers are indicated.