Extended Data Fig. 1: Analysis and purification of the SAMTom40-early intermediate.
From: A multipoint guidance mechanism for β-barrel folding on the SAM complex
a, Wild-type (WT) mitochondria were incubated with radiolabeled WT Tom40 and Tom40G354A mutant precursor proteins (pulse), re-isolated and incubated to follow the assembly (chase) of the precursor proteins by blue native-PAGE and radioimaging. Lane 8 shows a higher contrast image of lane 7 to visualize the assembly into the mature TOM complex. b, Digitonin lysed mitochondria isolated from WT and Tom40G354A strains grown at 23 °C in YPG were analyzed by blue native-PAGE and immunoblotting against Tom22. c, Radiolabeled wild-type (WT) Tom40 and Tom40G354A precursor proteins were imported into isolated WT mitochondria and those with Sam50 lacking the POTRA domain (ΔPOTRA), Tom5 (Δ5), Tom6 (Δ6), Tom7 (Δ7), or Sam37 (Δ37) for 30 min at 25 °C, and solubilized complexes were analyzed by blue-native PAGE and radioimaging. TOM, mature TOM complex; SAM-Tom40, SAMTom40-early and/or SAMTom40-folded, SAM-Tom40-5-6, SAMTom40-5-6, and Tom40-5-6-7, the assembly intermediate containing Tom40, Tom5, Tom6, and Tom7. d, [35S]Tom40G354A precursor was imported into wild-type (WT) and sam37Δ mitochondria for the indicated periods. Where indicated, recombinant Sam37 was imported into sam37Δ mitochondria before [35S]Tom40G354A was imported. The samples were lysed with digitonin and analyzed by native electrophoresis and radioimaging. e, The elution profile of gel filtration of the purified SAMTom40-early intermediate in 0.02% GDN. f, g, SDS-PAGE (f) and blue-native PAGE (g) gels of the SAMTom40-early intermediate stained with Coomassie Brilliant Blue (CBB). Data are representative of two (panels a-c), four (panel d), or five times (f, g) independent experiments.
