Tom70 is necessary for PINK1 import.

<p>(A) Mitochondria isolated from mouse liver were treated at 0°C for 10 min with 50 µg/ml trypsin and then analyzed by western blotting using antibodies against Tom70 (OMM) and Mia40 (IMS) proteins. (B) Radiolabeled PINK1 was incubated at 15°C with intact (Tryp−) or trypsin treated (Tryp+) mitochondria for the indicated time periods. Samples were then analyzed by SDS-PAGE and autoradiography. (C) Bands corresponding to the mature imported protein were quantified. The amount of mature PINK1 at 15 min import into intact mitochondria (Tryp−) was set to 100%. The results are the average of three independent experiments ± SD. (D) The membrane potential of intact mitochondria (Tryp−), trypsin treated mitochondria (Tryp+) and intact mitochondria treated with 1 µM CCCP and 1 µM valinomycin were measured by uptake of fluorescent dye diSC3(5). The results are the average of three experiments ± SD. (E, F) Mitochondria isolated from control cells (−Dox) and fromTom22 (E, +Dox) or 70 knockdown cells (F, +Dox) were analyzed by western blotting using the indicated antibodies. (G, H) Radiolabeled PINK1 was incubated at 15°C for the indicated time intervals with mitochondria isolated from control (−Dox), Tom22-depleted (G, +Dox), or Tom70 depleted cells (H, +Dox). Samples were analyzed by SDS-PAGE and autoradiography (upper panels). Bands corresponding to the mature imported protein were quantified. The amount of mature PINK1 after 30 min import of control mitochondria (−Dox) was set to 100% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058435#pone-0058435-g004" target="_blank">Fig. 4G</a>, H lower panels). Graphs represent mean values of three independent experiments ± SD.