Extended Data Fig. 4.: Structural characterization of the Tom40-early folding intermediate on SAM.
From: A multipoint guidance mechanism for β-barrel folding on the SAM complex
a, Interactions between loop 1 (between β1 and β2) and the β14-17 region of Sam50 in the SAMdimer complex (PDB ID: 7BTW). b, Comparison of the structures of the Tom40 in the SAMTom40-early intermediate (red) and in the mature TOM complex (pink). c, HA-tagged cysteine-free (Cfree) Sam50 mutants with cysteine residues introduced at the indicated positions (β1 and β16) were expressed in yeast cells lacking endogenous Sam50. Mitochondria were isolated and treated with 1 mM CuSO4 (oxidant) or 1 mM DTT (reductant) and proteins were analyzed by non-reducing SDS-PAGE followed by immunoblotting with the indicated antibodies. Ox, oxidized form with disulfide-bond formation; Red, reduced form. d, Mitochondria treated with or without CuSO4 (oxidant) were solubilized with digitonin and mixed without (Mito) or with GST bound to glutathione-Sepharose beads (GST) or GST followed by the Tom40 β-signal peptide fused with GST bound to glutathione-Sepharose beads (GST-β). The GST domain contains the C-terminally attached cleavage site for 3 C protease. Then Sam50 bound to the Tom40 β-signal was eluted from the beads by 3 C protease treatment, and proteins were analyzed by non-reducing SDS-PAGE followed by immunoblotting with the anti-HA antibodies. e, Radiolabeled Por1β1-19,C276 (19 β-strands) and Por1β2-19,C276 (18 β-strands) were imported for 30 min at 25 °C into Sam50Cfree and Sam50C130 mitochondria. Subsequently, samples were oxidized with 4-DPS and analyzed on non-reducing SDS-PAGE and radioimaging. Arrowhead, disulfide-bond of Sam50 and Por1 precursor; circle, porin precursor. f–i, Cysteine-free Tom40 (CF) and the indicated double-cysteine mutants Tom40 mutants associated with the SAM complex and those integrated into the mature TOM complex were isolated by FLAG purification (g, i) and HA purification (f, h), respectively, as in Fig. 3a. Proteins were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. L, load (5%); E, elute (50%). Data are representative of three (panel c–i) independent experiments.
