Fig. 3.
Pam18 affects lateral insertion of membrane-targeted precursors. a Schematic presentation of b2(220)-DHFR precursor. MTS, mitochondrial targeting signal; MPP, mitochondrial processing peptidase cleavage site; IMP, inner membrane protease cleavage site; HBD, heme-binding domain. b [35S]-labeled b2(220)-DHFR was imported as described in Fig. 2. c Quantification of intermediate (upper) and mature (lower) form of b2(220)-DHFR from b. d Quantification of the b2(220)-DHFR import after 15 min from b (processed = i + m); i, intermediate, m, mature; tim17∆+T17-P18 tim17∆ + TIM17–PAM18. Results are shown as mean ± SEM, n = 3. e Import of [35S]-labeled b2(220)-DHFR into Tim17–Pam18 and Tim17-TEV–Pam18 (TEV cleavage site was introduced between Tim17 and Pam18). Mitochondria were swollen and TEV protease treated. Subsequently, precursors were imported for 15 min. Samples were analyzed by SDS-PAGE and digital autoradiography and western blotting. Results are shown as mean ± SEM, n = 3. tim17∆+T17-P18 tim17∆/pam18∆ + TIM17–PAM18, AR, autoradiography; WB, western blot. f After import of [35S]-labeled Atp4 (membrane protein) and Atp5 (soluble matrix protein), samples were analyzed by blue-native PAGE/SDS-PAGE and subjected to digital autoradiography. g Quantification from e, upper panel, imported precursor after 30 min (SDS-PAGE), lower panel assembly efficiency normalized to import. Results are shown as mean ± SEM, n = 4