Fig. 3

Mpc2 and Mpc3 are imported by TIM22 and are independent of TIM23. a Wild-type (WT) and tim17-5 mitochondria, which display a specific defect in TIM23-mediated matrix import [46, 47], were heat-shocked for 10 min at 37 °C prior to import of radiolabeled Mpc2 or Mpc3 at 25 °C. Samples were analyzed by BN-PAGE and autoradiography. Quantification of import and assembly efficiency; the efficiency into WT mitochondria after 30 min was set to 100% (control), n = 3; error bars: SEM. b As a control, the matrix protein F₁β was imported into heat-shocked WT and tim17-5 mitochondria. Samples were analyzed by SDS-PAGE and autoradiography. p, precursor; m, mature form. c Mpc2 and Mpc3 were imported at 25 °C into heat-shocked WT mitochondria and tim17-4 mitochondria that display a defect in TIM23-mediated sorting into the inner membrane [46, 47]. Samples were analyzed and quantitated as in a; n = 3; error bars: SEM. d As a control, the IM sorting substrate b₂(220)-DHFR was imported into heat-shocked WT and tim17-4 mitochondria. Samples were analyzed by SDS-PAGE and autoradiography. i, intermediate form; m, mature form. e Mpc2 was imported at 25 °C into mitochondria from WT and TIM22-specific yeast mutant strains, tim18Δ or tim22-14, and analyzed by BN-PAGE and autoradiography. Quantification of import and assembly efficiency as in a; n = 3; error bars: SEM. f Mpc3 was imported at 25 °C into mitochondria from WT, tim18Δ and tim22-14 strains as in e. Quantification of import and assembly efficiency as in a; n = 3; error bars: SEM. g The model carrier substrate AAC was imported at 25 °C into tim18Δ and tim22-14 mitochondria (left panel) and analyzed as the Mpc2/Mpc3 import reactions. As a control, the matrix-targeted precursor of F₁β was imported into these mitochondria (right panel) and analyzed by SDS-PAGE and autoradiography. m, mature form. In all experiments, non-imported precursors were degraded with PK