Extended Data Fig. 8: Role of the α-protrusion of Sam37 in Tom40 folding on the SAM complex.
From: A multipoint guidance mechanism for β-barrel folding on the SAM complex
a, b, Behaviour of the variant precursor Tom40M94L (used in Fig. 5e). Radiolabeled Tom40 constructs were analyzed by SDS-PAGE and radioimaging (a). The variant precursor Tom40M94L (shown in lane 1) prevents the generation of a truncated Tom40 via translation initiation from the internal methionine M94. Radiolabelled wild-type (WT) Tom40 and Tom40M94L precursor proteins were imported into isolated WT mitochondria for the indicated periods (min) at 25 °C, and solubilized complexes were analysed by blue-native PAGE and radioimaging (b). 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. Tom40M94L assembled like WT Tom40. c–i, Analysis of the α-protrusion of Sam37. c, Growth of YPH499 wild type, Sam37Δ186-209 and sam37Δ yeast cells on fermentable (YPD) and non-fermentable (YPG) medium at 19 °C, 24 °C, 30 °C and 36 °C °C (Sam37Δ186-209 is chromosomally expressed and contains a GAG linker at the site of truncation). d, Steady-state protein levels of isolated mitochondria from YPH499 wild type and Sam37Δ186-209 cells, as analyzed by Tris/Tricine gel electrophoresis and immunoblotting. Mitochondria were isolated on non-fermentable (YPG) medium at 23 °C. e, f, Radiolabeled Tom22 (e) and Tom20 (f) precursor proteins were imported into isolated YPH499 wild type, Sam37Δ186-209, and sam37Δ mitochondria for the indicated periods, and the digitonin-solubilized complexes were analyzed by blue native PAGE and autoradiography. TOM, the TOM complex. g–i, Radiolabeled Tom5, Tom6, and Tom7 precursor proteins were imported into isolated YPH499 wild type and Sam37Δ186-209 mitochondria for the indicated periods at 25 °C, and the digitonin-solubilized complexes were analyzed by blue native PAGE and radioimaging. TOM, the TOM complex; TOM40-5-6-7, the assembly intermediate consisting of Tom40, Tom5, Tom6, and Tom7. Data are representative of two (panels a, b, d, g–i) or three (panels c, e, f) independent experiments.
