Molecular architecture of the active mitochondrial protein gate by Takuya Shiota, Kenichiro Imai, Jian Qiu, Victoria L. Hewitt, Khershing Tan, Hsin-Hui Shen, Noriyuki Sakiyama, Yoshinori Fukasawa, Sikander Hayat, Megumi Kamiya, Arne Elofsson, Kentaro Tomii, Paul Horton, Nils Wiedemann, Nikolaus Pfanner, Trevor Lithgow, and Toshiya Endo Science Volume 349(6255):1544-1548 September 25, 2015 Published by AAAS

Fig. 1 The pore interior of the β barrel of Tom40 is the protein import channel for preproteins. The pore interior of the β barrel of Tom40 is the protein import channel for preproteins. (A) In vivo photo–cross-linking of BPA at the indicated positions of Tom40 with Tom22 analyzed by SDS-PAGE followed by immunoblotting (top) and quantification (bottom). (B and C) [35S]pSu9-DHFR (Su9 presequence fused to dihydrofolate reductase) (B) or [35S]AAC-DHFR (ADP-ATP carrier fused to DHFR) (C) was incubated with mitochondria [pretreated with valinomycin for (B)] with BPA-bearing Tom40 at 4°C (B) or 25°C in the presence of 1 μM methotrexate and 1 mM NADPH (C), for 10 min, and UV-irradiated. Affinity-purified cross-linked products (asterisk) were subjected to SDS-PAGE and radioimaging. Variation in the apparent molecular weight of the cross-linked products may reflect different configurations. β-strand numbers and BPA positions (red and green, side chain facing the pore interior or membrane, respectively) are indicated. The cross-links of the neighboring residues 356 (weak) and 357 (strong) with Tom22 may suggest imperfections in the last β strand 19 around this position. p, precursor form; i, processing intermediate form; m, processed mature form. (D) Quantification of the cross-linked products in (B) and (C). (E) The side chains of the Tom40 residues cross-linked to pSu9-DHFR or AAC-DHFR are shown with color reflecting the amount of cross-linked products detected. (F) The acidic patches (red) and hydrophobic patches (green) are shown in the pore interior of Tom40, in relation to the position of Tom22TM (Fig. 4, inset). Takuya Shiota et al. Science 2015;349:1544-1548 Published by AAAS

Fig. 2 The N-terminal segment of Tom40 interacts with elements of the carrier protein import pathway. The N-terminal segment of Tom40 interacts with elements of the carrier protein import pathway. (A) Topology model and BPA cross-linking results of Tom40. Circles, pentagons, and squares indicate residues in the loop, α helix, or β strand, respectively. BPA-incorporated residues are labeled in bold. Cross-linked partners are indicated by different colors. (B) UV-dependent in vivo photo–cross-linking of BPA at the indicated position of Tom40 detected by immunoblotting (top) and quantification (bottom). (C and D) In vitro import of [35S]labeled presequence-containing (C) and presequence-less (D) preproteins into the tom40 mutant mitochondria at 25°C. After proteinase K treatment, imported proteins were analyzed by SDS-PAGE and radioimaging. The imported amount at the longest incubation time was set to 100% (control). Values are mean ± SEM (n = 3). (E) Serial dilutions of the Tom40 N-terminal truncation mutant cells with overexpression of the indicated proteins were spotted on SCD (-Ura, -Trp) (glucose), SCGal (-Ura, -Trp) (galactose), and SCGly + 0.05% d-glucose (-Ura, -Trp) (glycerol + galactose) media and grown at 30°C for 3 days (glucose and galactose) or 4 days (glycerol + galactose). WT, corresponding TOM40-(His)10 strain; PiC, phosphate carrier; DiC, dicarboxylate carrier. Takuya Shiota et al. Science 2015;349:1544-1548 Published by AAAS

Fig. 3 The native TOM complex dynamically exchanges with the Tom40 dimeric complex. The native TOM complex dynamically exchanges with the Tom40 dimeric complex. (A) Mitochondria with the indicated Tom40 Cys residues were treated with (+) or without (–) the cross-linker (1,4-bismaleimidobutane) [BMB (XL)]. Proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to Tom40. Cross-linked products are indicated by the asterisk and arrowheads. (B) Schematic models for (A). (C) (Top) The Tom40 β barrel showing the residues cross-linked with Tom22. (Botom) Tom22-interacting regions of Tom40 (blue and yellow) with the Tom22-cross-linked residues (pink circles). (D) Tom40-interacting regions of Tom22TM (orange and green) (18). (E) In vivo cross-linking of BPA in Tom40 and Tom22 was detected by immunoblotting. Cross-linked products are indicated. (F) Schematic models for (E). (G) Mitochondria with the Tom40 Cys mutations were treated with (+) or without (–) the cross-linker BMOE [bis(maleimido)ethane]. Proteins were analyzed by SDS-PAGE (lanes 1 to 4), BN-PAGE (lanes 5 and 6), or 2D-PAGE (first- BN-PAGE and second-dimensional SDS-PAGE) and immunoblotting. Cross-linked products are indicated by the asterisk and arrowheads. Takuya Shiota et al. Science 2015;349:1544-1548 Published by AAAS

Fig. 4 Subunit organization of the TOM complex. Subunit organization of the TOM complex. Subunit arrangement of the Tom40 β barrel and TM α helices of Tom5, Tom6, Tom7, and Tom22, based on the BPA cross-linking results and the proposed model for the exchange of the Tom40-Tom22 trimeric complex with the Tom40 dimer. The inset shows possible interactions between Tom22TM and the Tom40 β barrel, with key residues suggested from fig. S8. Basic residues, acidic residues, and others are colored in blue, red, and pink, respectively. The Tom22TM α helix, possibly bent at Pro112, tethers two Tom40 molecules through the interactions of its N-terminal and C-terminal parts with conserved residues of adjacent Tom40 molecules. Takuya Shiota et al. Science 2015;349:1544-1548 Published by AAAS