Figure 2.

Tom40 interacts with Tom20 on its insertion pathway. (A) Radiolabeled Tom40 precursor was incubated with isolated mitochondria for 15 min at 0°C or 25°C. The mitochondria were reisolated, and divided into three aliquots. One aliquot was left on ice (−) while to the other two, the chemical cross-linkers DSG or S-MBS, were added for 40 min on ice. The cross-linking reagents were quenched with 80 mM glycine, pH 8.0, and the mitochondria were pelleted and loaded on SDS-PAGE. The gel was analyzed by autoradiography for detection of imported protein or immunostained with antibodies against Tom40 to detect the endogenous Tom40. (B) Identification of the Tom40-Tom20 adduct by immunoprecipitation. Radiolabeled Tom40 was incubated with isolated mitochondria for 15 min at 0°C. After reisolation, the cross-linker S-MBS was added (see above), and mitochondria were pelleted and solubilized with SDS- and Triton X-100–containing buffer. Aliquots were subjected to immunoprecipitation with antibodies against Tom20 or antibodies derived from preimmune serum. The immunoprecipitates were solubilized in sample buffer and analyzed as above.