Fig. 4.

Pam18 regulates the lateral gate. a Yeasts were shifted to glycerol medium and grown for 4 h (Mic10 and Atp20) or 6 h (all other tested proteins). Cell lysates were analyzed for levels of indicated outer membrane (OM), matrix or inner membrane (IM) proteins by western blot. b Quantification of blots from a. Amounts of proteins in percentage of WT control. Results are shown as mean ± SEM, n = 3 (Mic10 and Atp20) n = 4 for all others. c, d Import of [³⁵S]-labeled b₂(220)-DHFR in mgr2∆ and pam17∆ and corresponding wild-type as described in Fig. 2. e WT mitochondria or mitochondria containing Pam18^His or ^HisS*Tim23 were solubilized in digitonin buffer and subjected to Ni-NTA affinity chromatography. Samples were analyzed by SDS-PAGE and western blotting. HisS*, His-SUMOstar. f After import of [³⁵S]-labeled b₂(220)-DHFR without proteinase K treatment, mitochondria were solubilized in digitonin buffer and subjected to anti-Tim23 immunoisolation. Samples were analyzed by SDS-PAGE, western blotting, and digital autoradiography. AR, autoradiography; WB, western blot. g Quantification of co-isolated i-b₂(220)-DHFR normalized to isolated Tim23. Results are shown as mean ± SEM, n = 4; p, precursor; I, intermediate;m, mature