Figure 2.

Bim_S localizes to and inserts into mitochondrial membranes in HeLa cells and in isolated mitochondria. (a) Mitochondria (m) from T-REx-HeLa cells (A2 clone) uninduced or tet-induced as indicated were isolated and separated alongside the soluble cytosolic fractions (c) on 12.5% SDS-PAA gels. CoxIV is a marker for mitochondria, caspase-8 for cytosolic proteins. (b) Isolated mitochondria were treated with 0.1 M Na₂CO₃ to separate membrane inserted (Ins.) from soluble (Att., membrane attached, intermembrane space and matrix) fractions. Both fractions were analyzed by Western blotting for Bim, CoxIV as a marker for mitochondrial membranes and Bax. (c) Import of in vitro–translated Bim_EL or Bim_S into isolated HeLa mitochondria. Radiolabeled precursors of Bim_EL and Bim_S were incubated for 10 and 30 min at 30°C with mitochondria isolated from HeLa cells. Subsequently, the samples were split. One third was treated with 50 μg/ml proteinase K (PK) for 20 min on ice, another one was left untreated. The last third was subjected to alkaline extraction resulting in a pellet fraction (P; containing integral membrane proteins) and a supernatant fraction (S; containing soluble and peripherally attached proteins). For comparison, 10% of the total input of radiolabeled precursors was included (T). Mitochondria were reisolated and import was analyzed by SDS-PAGE and autoradiography. ^*, very likely proteolysis products.